Composition and method for treating lupus nephritis

ABSTRACT

The present invention provides novel isolated BFLP0169 polynucleotides and polypeptides encoded by the BFLP0169 polynucleotides. Also provided are the antibodies that immunospecifically bind to a BFLP0169 polypeptide or any derivative (including fusion derivative), variant, mutant or fragment of the BFLP0169 polypeptide, polynucleotide or antibody. The invention additionally provides methods in which the BFLP0169 polypeptide, polynucleotide and antibody are utilized in the detection and treatment of a broad range of pathological states, as well as to other uses.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/719,385, filed Nov. 21, 2003 now U.S. Pat. No. 7,060,797,which claims priority to U.S. provisional patent application 60/428,094,filed Nov. 21, 2002. The entire contents of these applications areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates generally to nucleic acids and polypeptides andmore specifically to nucleic acids and polypeptides encodingpolypeptides useful for detecting and treating lupus nephritis, as wellas for identifying therapeutic agents for treating the same.

BACKGROUND OF THE INVENTION

Lupus nephritis is an example of a “classical” auto-immune disease inwhich the patient's immune system attacks his/her own organs. It hasbeen estimated that 45-75% of lupus patients eventually suffer from someform or other of kidney damage. Lupus varies greatly in severity frommild cases requiring minimal intervention to those in which significantdamage occurs to vital organs such as lungs, kidneys, heart and brain,and which ultimately can be fatal. Lupus is predominantly a femaledisease, with an approximate female to male ratio being 9:1. In NorthAmerica, it is estimated to affect 1 in 500 females mainly between theage of 20 to 40 years.

There is no known cure for lupus. Treatment is typically directed atcontrolling the symptoms with the hope of putting the disease intoremission. Recently, the antibiotic rapamycin has been demonstrated tobe an effective therapy in treating lupus nephritis in a murine model ofthe disease.

SUMMARY OF THE INVENTION

The invention is based, in part, upon the discovery of a gene, namedBFLP0169, whose expression is increased in kidney tissue in mice withlupus nephritis; however, the expression level of the gene does notdecrease markedly in response to treatment with rapamycin. Thisexpression profile indicates that the product of the BFLP0169 geneinteracts with rapamycin when this antibiotic is administered toameliorate the symptoms of lupus nephritis. In the absence of rapamycin,the gene product is free to bring about the diseased state, and itseffects can include the activation of genes required to bring about thediseased state. In the presence of rapamycin, the BFLP0169 gene productis inactive and the diseased state diminishes. Accordingly, the BFLP0169protein is useful as a target for identifying agents that, likerapamycin, are useful in treating symptoms of lupus nephritis.

In one aspect, the invention provides an isolated nucleic acid moleculethat includes the sequence of a nucleotide sequence encoding a BFLP0169gene product. In a preferred embodiment, the nucleotide sequenceincludes the sequence of SEQ ID NO:1, or a fragment, homolog, analog orderivative thereof. The nucleic acid can include, e.g., a nucleic acidsequence encoding a polypeptide at least 70%, e.g., 80%, 85%, 90%, 95%,98%, or even 99% or more identical to a polypeptide that includes theamino acid sequences of SEQ ID NO:2. The nucleic acid can be, e.g., agenomic DNA fragment, or a cDNA molecule.

Also included in the invention is a vector containing one or more of thenucleic acids described herein, and a cell containing the vectors ornucleic acids described herein.

The invention is also directed to host cells transformed with a vectorcomprising any of the nucleic acid molecules described above.

In another aspect, the invention includes a pharmaceutical compositionthat includes a BFLP0169 nucleic acid and a pharmaceutically acceptablecarrier or diluent.

In a further aspect, the invention includes a substantially purifiedBFLP0169 polypeptide, e.g., any of the BFLP0169 polypeptides encoded bya BFLP0169 nucleic acid, and fragments, homologs, analogs, andderivatives thereof. The invention also includes a pharmaceuticalcomposition that includes a BFLP0169 polypeptide and a pharmaceuticallyacceptable carrier or diluent.

In a still further aspect, the invention provides an antibody that bindsspecifically to a BFLP0169 polypeptide. The antibody can be, e.g., amonoclonal or polyclonal antibody, and fragments, homologs, analogs, andderivatives thereof. The invention also includes a pharmaceuticalcomposition including BFLP0169 antibody and a pharmaceuticallyacceptable carrier or diluent. The invention is also directed toisolated antibodies that bind to an epitope on a polypeptide encoded byany of the nucleic acid molecules described above.

The invention also includes kits comprising in one or more containersone or more of a compound that is a BFLP0169 nucleic acid, a BFLP0169polypeptide and/or an antibody to a BFLP0169 polypeptide. The kit ispreferably provided with instructions for use. If desired, the compoundsin the kits are provided along with a pharmaceutically acceptablecarrier.

The invention further provides a method for producing a BFLP0169polypeptide by providing a cell containing a BFLP0169 nucleic acid,e.g., a vector that includes a BFLP0169 nucleic acid, and culturing thecell under conditions sufficient to express the BFLP0169 polypeptideencoded by the nucleic acid. The expressed BFLP0169 polypeptide is thenrecovered from the cell. Preferably, the cell produces little or noendogenous BFLP0169 polypeptide. The cell can be, e.g., a prokaryoticcell or eukaryotic cell.

The invention is also directed to methods of identifying a BFLP0169polypeptide or nucleic acid in a sample by contacting the sample with acompound that specifically binds to the polypeptide or nucleic acid, anddetecting complex formation, if present.

The invention further provides methods of identifying a compound thatmodulates the activity of a BFLP0169 polypeptide by contacting aBFLP0169 polypeptide with a compound and determining whether theBFLP0169 polypeptide activity is modified.

The invention is also directed to compounds that modulate BFLP0169polypeptide activity identified by contacting a BFLP0169 polypeptidewith the compound and determining whether the compound modifies activityof the BFLP0169 polypeptide, binds to the BFLP0169 polypeptide, or bindsto a nucleic acid molecule encoding a BFLP0169 polypeptide.

In another aspect, the invention provides a method of determining thepresence of or predisposition of a BFLP0169-associated disorder in asubject. The method includes providing a sample from the subject andmeasuring the amount of BFLP0169 polypeptide in the subject sample. Theamount of BFLP0169 polypeptide in the subject sample is then compared tothe amount of BFLP0169 polypeptide in a control sample. An alteration inthe amount of BFLP0169 polypeptide in the subject protein samplerelative to the amount of BFLP0169 polypeptide in the control proteinsample indicates the subject has a tissue proliferation-associatedcondition. A control sample is preferably taken from a matchedindividual, i.e., an individual of similar age, sex, or other generalcondition but who is not suspected of having a tissueproliferation-associated condition. Alternatively, the control samplemay be taken from the subject at a time when the subject is notsuspected of having a tissue proliferation-associated disorder. In someembodiments, the BFLP0169 is detected using a BFLP0169 antibody.

In a further aspect, the invention provides a method of determining thepresence of or predisposition of a BFLP0169-associated disorder in asubject. The method includes providing a nucleic acid sample, e.g., RNAor DNA, or both, from the subject and measuring the amount of theBFLP0169 nucleic acid in the subject nucleic acid sample. The amount ofBFLP0169 nucleic acid sample in the subject nucleic acid sample is thencompared to the amount of a BFLP0169 nucleic acid in a control sample.An alteration in the amount of BFLP0169 nucleic acid in the samplerelative to the amount of BFLP0169 in the control sample indicates thesubject has a tissue proliferation-associated disorder.

In a still further aspect, the invention provides a method of treatingor preventing or delaying a BFLP0169-associated disorder. The methodincludes administering to a subject in which such treatment orprevention or delay is desired a BFLP0169 nucleic acid, a BFLP0169polypeptide, or a BFLP0169 antibody in an amount sufficient to treat,prevent, or delay a tissue proliferation-associated disorder in thesubject. Examples of such disorders include rheumatoid arthritis andmultiple sclerosis.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a histogram showing relative levels of gene expression in themouse ortholog of the human BFLP0169 gene in NZB×NZWF1 kidneys before,during, and after rapamycin treatment, as well as in various controlmouse strains and conditions.

DETAILED DESCRIPTION OF THE INVENTION

The BFLP0169 nucleic acid sequences disclosed herein were identifiedbased on changes in expression of the gene in kidneys of a lupusnephritis model mouse as compared to expression of the gene in kidneysfrom non non-diseased mice. More particularly, the gene is expressed atrelatively low levels in young mice and mice that do not show symptomsof lupus nephritis. Gene expression is elevated in mice with lupusnephritis, and is lower in mice that have been successfully treated withrapamycin or anti-B7 antibodies. The observation that expression levelsreturn to normal when kidney function is normal indicates that elevatedlevels are related to, and diagnostic of, disease progression. Blockingthe function of these genes may inhibit or retard disease progression.Expression levels can also to used to assess and compare effectivenessof various therapeutic interventions.

Accordingly, the BFLP0169 nucleic acid sequences are useful fordetecting the presence of lupus nephritis in a subject. Elevated levelsof BFLP0169 transcripts orpolypeptides relative to levels in controlsamples indicate the presence of lupus nephritis in the subject.BFLP0169 nucleic acid sequences can also be used to monitor theeffectiveness of treatments for lupus nephritis: a decrease inexpression of BFLP0169 genes relative to levels in diseased treatmentsdemonstrates that the treatment is effective.

The BFLP0169 sequences can additionally be used to identify therapeuticagents for treating or preventing lupus nephritis in a subject. Forexample, a BFLP0169 polypeptide can be contacted with a test agent.Binding of the BFLP0169 polypeptide to the test agent reveals that thetest agent modulates BFLP0169 activity. The BFLP0169-binding agent canbe further tested to determine if it acts to promote or inhibit lupussymptoms in a test organism (e.g., a NZB×NZW mouse). Inhibition of lupussymptoms reveals that the agent is useful for treating or preventinglupus nephritis, or symptoms associated with lupus nephritis. Additionalutilities are disclosed herein.

A 5987 nucleotide sequence that includes a human BFLP0169 nucleic acidis shown in Table 1 (SEQ ID NO:1). The human sequence was identified asthe human ortholog of a murine gene whose expression is increased in aNZB×NZW mouse with lupus nephritis-like symptoms.

Nucleotides 1-5259 of the sequence shown in Table 1 encode a polypeptideof 1753 amino acids, whose sequence is shown in Table 2 (SEQ ID NO:2).

TABLE 1 ATGATCAGAAAGAGCAAAATTACCTCTGTTCTCTCATTTTGCAGGAGCAGTAGAGAACTGTGGACTATTCTGCTTGGAAGGTCAGCTCTGAGAGAGCTGAGTCAGATTGAGGCAGAACTGAATAAACATTGGCGGCGATTGTTAGAGGGGCTTTCTTACTACAAACCTCCCAGTCCAAGTTCAGCTGAAAAAGTGAAAGCTAATAAAGATGTAGTTCACCATTGAAGGAACTGGGTTTAAGAATCAGCAAGTTTTTGGGTCTTGATGAAGAACAGAGTGTGCAGTTACTCCAGTGTTACCTGCAAGAGGACTACAGGGGTACTCGGGACTCAGTAAAGACAGTACTGCAAGATGAGAGGCAGAGCCAGGCCTTAATCCTGAAGATTGCAAGATTATTATTATGAAGAAGAACCTGTATTCTTCGTTGTGTCTTACACCTTCTCACTTACTTCCAAGATGAAAGACACCCCTATAGGGTTGAATATGCAGACTGTGTTGATAAATTGGAGAAGGAACTAGTTTCAAAATACAGACAGCAGTTCGAAGAGCTTTATAAAACTGAAGCACCAACTTGGGAGACACATGGAAATCTCATGACAGAGCGCCAAGTGTCTCGCTGGTTTGTTCAGTGCCTTCGGGAACAGTCCATGCTGCTAGAAATTATTTTCCTTTATTATGCATACTTTGAGATGGCACCCAGTGACTTACTTGTATTACCAAGATGTTTAAAGAGCAAGGATTTGGTAGTAGGCAGACCAATAGGCACCTGGTGGATGAGACTATGGATCCTTTTGTAGATCGGATTGGCTACTTCAGTGCCCTCATCCTGGTGGAGGGCATGGATATCGAGTCCTTGCATAAGTGTGCTTTGGATGACAGAAGAGAACTGCATCAGTTTGCGCAGGATGGGCTTATTTGTCAGGATATGGACTGTTTAATGTTGACCTTTGGGGACATTCCACATCATGCCCCAGTGCTTTTGGCCTGGGCTCTCCTCCGTCACACTCTGAACCCAGAAGAGACAAGCAGTGTGGTCCGGAAGATAGGTGGCACAGCCATCCAGCTGAATGTGTTTCAGTACTTGACCCGATTGCTCCAGTCCCTTGCCAGTGGGGGAAATGATTGCACCACCAGCACTGCATGCATGTGTGTCTATGGACTGCTCTCTTTCGTTCTGACCTCGTTGGAGCTGCACACCCTGGGCAATCAGCAGGATATAATTGATACAGCATGTGAAGTATTGGCCGACCCTTCTCTTCCGGAACTGTTCTGGGGAACAGAGCCAACTTCTGGCCTTGGGATCATTCTGGACAGTGTGTGTGGAATGTTTCCCCACCTTCTCTCCCCACTCCTGCAACTGCTCCGAGCCCTGGTATCAGGGAAGTCCACAGCCAAAAAGGTGTATAGCTTCTTGGATAAGATGTCTTTCTACAATGAACTTTATAAACACAAGCCTCATGATGTGATCTCCCATGAAGATGGAACTCTTTGGCGGAGACAAACACCCAAACTCCTTTATCCCCTTGGGGGTCAAACCAACCTTCGCATACCTCAAGGCACTGTGGGCCAAGTAATGTTGGATGATAGGGCATACCTGGTACGCTGGGAATACTCCTATAGCAGCTGGACCCTCTTTACCTGCGAGATTGAAATGTTGCTTCATGTTGTTTCAACTGCAGATGTGATTCAGCACTGCCAGCGAGTCAAACCCATCATTGATCTCGTCCATAAGGTCATCAGTACAGACCTGTCGATAGCAGACTGTCTCCTGCCCATCACATCTCGCATCTACATGCTGCTGCAGCGGTTAACGACAGTGATCTCCCCACCTGTGGATGTCATTGCTTCTTGTGTCAACTGCTTAACTGTTTTGGCTGCCCGCAATCCAGCAAAGGTCTGGACTGATCTTCGTCACACAGGTTTTTTACCATTTGTGGCCCATCCTGTCTCCAGCCTGAGTCAGATGATTAGTGCGGAAGGGATGAATGCTGGAGGGTACGGAAACCTCTTGATGAACAGTGAACAGCCTCAGGGCGAGTATGGGGTTACTATTGCCTTTCTGCGCTTGATCACCACCCTTGTCAAGGGGCAACTTGGTAGTACCCAGAGCCAAGGACTTGTACCCTGTGTAATGTTTGTGCTGAAGGAGATGCTTCCCAGCTACCATAAGTGGCGCTACAACTCTCATGGAGTGAGGGAACAGATTGGTTGCCTGATCTTGGAGCTGATTCATGCGATACTGAACCTGTGCCACGAGACAGACCTGCACAGCAGTCATACTCCCAGCCTGCAGTTTCTCTGCATCTGCAGCCTGGCATACACAGAAGCAGGACAGACAGTTATCAATATCATGGGCATTGGCGTGGACACCATTGACATGGTGATGGCTGCTCAGCCTCGAAGTGATGGGGCAGAGGGCCAGGGGCAGGGCCAGCTGCTGATCAAGACAGTGAAACTGGCATTCTCCGTCACCAACAATGTTATTCGGCTGAAACCTCCTTCTAATGTGGTGTCCCCCCTGGAACAGGCTCTCTCACAACATGGTGCTCATGGAAACAACCTCATTGCTGTTCTAGCCAAATACATCTACCACAAACATGACCCTGCTTTGCCACGTCTTGCCATTCAGCTGCTGAAACGTCTGGCCACGGTGGCCCCAATGTCAGTGTATGCTTGTCTGGGCAATGATGCGGCTGCCATTCGTGATGCCTTCCTGACCCGATTGCAGAGCAAAATTGAGGACATGCGCATCAAAGTCATGATTCTAGAGTTCCTCACTGTTGCAGTAGAGACCCAGCCAGGCCTCATCGAACTGTTTCTGAACCTGGAAGTTAAGGATGGCAGTGATGGCTCAAAGGAATTCAGCCTTGGGATGTGGAGCTGTCTCCATGCAGTGCTGGAGCTGATTGATTCCCAACAGCAAGATCGATACTGGTGCCCACCCCTGCTGCATCGTGCCGCCATTGCCTTTTTGCATGCTCTGTGGCAGGATCGGAGGGACAGTGCCATGCTGGTCCTCCGAACCAAACCCAAGTTTTGGGAAAATTTAACCAGTCCGCTGTTTGGAACCCTTTCTCCTCCCTCTGACAAATCAGAGCCCAGCATCCTGGAAACCTGTGCCCTAATCATGAAGATAATTTGCTTGGAGATATACTATGTAGTAAAGGGTTCATTAGACCAGTCATTAAAGGATACACTGAAGAAATTTTCCATCGAGAAACGCTTTGCCTACTGGTCAGGGTATGTCAAGTCATTGGCAGTTCACGTGGCCGAAACAGAAGGCAGCAGCTGCACCTCCTTGTTAGAGTACCAGATGCTGGTGTCCGCCTGGAGGATGCTTCTCATCATTGCCACCACTCATGCAGATATAATGCACCTGACTGACTCTGTGGTGCGTCGCCAGCTCTTTCTTGACGTGCTTGATGGAACCAAAGCATTACTCCTAGTTCCAGCCTCAGTGAACTGCCTTCGCCTTGGCTCCATGAAGTGCACTCTGCTGCTTATCCTCCTCCGGCAGTGGAAGAGAGAGTTAGGTTCTGTGGATGAAATCCTTGGACCCTTGACGGAGATCCTGGAGGGAGTGCTGCAGGCCGACCAGCAACTCATGGAGAAGACCAAGGCCAAGGTGTTCTCAGCATTCATCACAGTGTTGCAAATGAAGGAGATGAAGTAAGTGACATCCCCCAGTACTCCCAGCTGGTGCTGAATGTCTGTGAGACCCTCCAAGAGGAAGTGATTGCACTCTTCGACCAGACCCGCCACAGTCTGGCATTAGGCAGTGCCACAGAGGACAAGGACAGCATGGAGACTGACGACTGTTCTCGGTCCCGGCACAGGGACCAGCGTGATGGGGTGTGTGTCCTGGGCCTGCACCTGGCCAAGGAGCTGTGTGAGGTAGACGAGGATGGTGACTCCTGGCTGCAGGTAACCCGCAGGCTCCCCATCCTACCCACCCTCCTCACCACTCTAGAGGTGAGCCTTCGCATGAAGCAGAACCTGCATTTCACTGAGGCCACATTGCATCTGCTCCTCACCCTGGCTCGCACTCAGCAGGGAGCCACAGCAGTGGCTGGAGCTGGCATCACCCAGAGCATTTGTTTGCCCCTTCTGAGTGTGTACCAGCTGAGCACCAACGGCACAGCACAGACACCTAGTGCCTCTCGGAAGTCCCTGGATGCCCCCTCTTGGCCAGGAGTCTACCGCCTGTCCATGTCCCTGATGGAGCAGCTGCTCAAACTCTGCGCTACAACTTCCTGCCTGAGGCCCTGGACTTCGTGGGTGTCCACCAGGAGCGGACCTTACAGTGCCTCAACGCAGTGAGGACAGTGCAGAGTCTGGCCTGCCTGGAGGAGGCGGACCACACCGTGGGTTTTATTCTGCAGCTCTCTAACTTCATGAAGGAGTGGCACTTCCACCTGCCTCAGCTCATGCGTGATATCCAGGTCAACCTGGGTTACTTGTGCCAGGCATGTACCTCTCTCCTGCACAGTCGAAAGATGCTGCAGCATTACTTACAGAACAAAAATGGGGATGGCCTCCCCTCAGCTGTTGCCCAGCGAGTCCAGAGGCCACCGTCTGCTGCTTCTGCTGCCCCCTCCTCCTCAAAGCAGCCCGCTGCTGACACAGAGGCATCAGAGCAGCAGGCCTTGCACACAGTCCAGTATGGCCTTCTCAAGATCCTCAGCAAGACGCTGGCAGCCCTGCGCCACTTCACCCCAGATGTCTGCCAGATTCTGCTGGATCAGTCCCTGGACCTTGCTGAATACAACTTCCTGTTTGCCCTGAGCTTTACCACTCCCACCTTTGACTCCGAAGTGGCCCCCTCCTTCGGGACCCTTCTGGCCACAGTGAATGTGGCCCTCAACATGCTTGGAGAGCTGGACAAGAAAAAGGAGCCCCTCACCCAGGCAGTGGGGCTCAGCACACAGGCAGAAGGGACCAGGACGTTAAAGTCCCTCCTGATGTTTACCATGGAAAACTGCTTCTACCTGCTCATCTCTCAGGCGATGCGGTACCTTAGGGACCCGGCTGTGCACCCCCGGGACAAACAGCGGATGAAGCAGGAGCTCAGCTCTGAGTTGAGCACGCTGCTGTCCAGCCTCTCGCGCTACTTCCGCCGGGGAGCCCCCAGCTCCCCTGCCACTGGTGTCCTCCCCTCGCCGCAGGGCAAGTCCACCTCTCTCTCCAAAGCCAGCCCTGAGAGTCAGGAGCCTCTGATCCAGTTGGTGCAGGCGTTTGTCCGGCATATGCAAAGATAGGGCAGTGCTGTTCTGCCCACCTACCCCTCTCCACCAGCCTACACTGCACCCTGGCTGGCAGGGGTGCTGCTGGCTGCTAGGGCCTATACAATGGAGGGCACCTCCTGTCACCCCCCTCCCGGAGTAGCCACGACTCCAGCCACCACCCACTGACGTTATTTTTATACTAGATGAAGAGGTCAACAGCAGGCATGGGGAGCCGAGTCTTCTGTGCTCAGGTCCTCACGCTGCAGACGCCCCCTAGAGGAACTTTCCTTCCTTTCCAGCATTCCCCACAGCACTGCCGGCCAGGGGAGAGGCGGCAGCCCAGCAGAGGGCTCTATGCACGGGTTTCAAACCTGTTTTCCACACTCTGTCTTTGCAGTTTTGGTAATTCTGTGGTCTATTTATACAGATATTAAAATCTTGTTTATAGACAGCTGTGTGATGTTTAACTTCAAAGCCCAGGGATGACAACGTGGCTCTCAGAACCTAGAAAACTCCCCTGGCCAGGCGCCTGGGAGTGGGGCTGCAGCCTCGGGGGAAGGCAGGTACTGATGGATGGCTAGTTCACCAGCATCTCCTCATTCCTGTCCTTGGGCTGAGGGTTTGGCTGGGTGGGCGCTGTCAGATATTCCCTTCCTTGGCCTGCGCTGGTCCTGTCCTTGACCCTGCTTTCATTGGCCCAGTGGGCTGAGCTCATCCCTGGGTGAGCCTTTCTTGAAGCTCTGTGCCTTCCTATTTAT (SEQ ID NO:1)

TABLE 2 MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR (SEQ ID NO:2)

BFLP0169-like nucleic acids and polypeptides of the invention (includingthose shown in Table 1) are referred to herein as “BFLP0169 ” nucleicacids and polypeptides.

A BFLP0169 nucleic acid, and the encoded polypeptide, according to theinvention are useful in a variety of applications and contexts.

BFLP0169 shows homolgy to other proteins as shown in the BLAST resultsdecribed in Table 3. KIAA0169, IMAGE: 3461492, and 3598686, and cDNA:FLJ21639 are all proteins encoded from partial reading frames (expressedsequence tags (ESTs)) found in genomic DNA. Because BFLP0169 hashomology to these proteins, it is also encoded from either an entireopen reading frame, or part of a larger open reading frame (EST).

TABLE 3 Blast Results for BFLP0169 Gene Index/ Protein/ Length IdentityPositives Identifier Organism (aa) (%) (%) Expect gi|1136397|dbj| Homosapiens 1745 1635/1739 1635/1739 0.0 D79991.1| mRNA for (94%) (94%)KIAA0169 protein, partial cds gi|22046118|ref| similar to 1767 1635/17431635/1743 0.0 XP_052725.6| KIAA0169 protein (93%) (93%) (XM_052725)[Homo sapiens] gi|23618434|ref| similar to 1111  949/1111  982/1111 0.0XP_130085.2| KIAA0169 protein (85%) (87%) (XM_130085) [Homo sapiens]gi|13529308|gb| Unknown (protein 853 740/801 740/801 0.0AAH05407.1|AAH05407 for (92%) (92%) (BC005407) IMAGE: 3461492) [Homosapiens] gi|19343754|gb| Similar to 525 411/522 422/522 0.0 AAH25526.1|KIAA0169 protein (78%) (80%) (BC025526) [Mus musculus]

Table 4 shows a ClustalW alignment of BFLP0169 (SEQ ID NO:2) against theproteins described above in Table 3.

TABLE 4 ClustalW Analysis of SEQ ID NO: 2 1) SEQ ID NO: 2 2)gi|1136397|dbj|D79991.1| (SEQ ID NO: 21) 3)gi|22046118|ref|XP_052725.6| (XM_052725) (SEQ ID NO: 22) 4)gi|23618434|ref|XP_130085.2| (XM_130085) (SEQ ID NO: 23) 5)gi|13529308|gb|AAH05407.1|AAH05407 (BC005407) (SEQ ID NO: 24) 6)gi|19343754|gb|AAH25526.1| (BC025526) (SEQ ID NO: 25)

Residues 1-14 of SEQ ID NO:2 are referred to herein as SEQ ID NO:20. Thefragment of SEQ ID NO:21 that includes amino acids 1-6 is referred toherein as SEQ ID NO:26.

BFLP0169 Nucleic Acids

The nucleic acids of the invention include those that encode a BFLP0169polypeptide or protein. As used herein, the terms polypeptide andprotein are interchangeable.

In some embodiments, a BFLP0169 nucleic acid encodes a mature BFLP0169polypeptide. As used herein, a “mature” form of a polypeptide or proteindescribed herein relates to the product of a naturally occurringpolypeptide or precursor form or proprotein. The naturally occurringpolypeptide, precursor or proprotein includes, by way of nonlimitingexample, the full length gene product, encoded by the correspondinggene. Alternatively, it may be defined as the polypeptide, precursor orproprotein encoded by an open reading frame described herein. Theproduct “mature” form arises, again by way of nonlimiting example, as aresult of one or more naturally occurring processing steps that may takeplace within the cell in which the gene product arises. Examples of suchprocessing steps leading to a “mature” form of a polypeptide or proteininclude the cleavage of the N-terminal methionine residue encoded by theinitiation codon of an open reading frame, or the proteolytic cleavageof a signal peptide or leader sequence. Thus a mature form arising froma precursor polypeptide or protein that has residues 1 to N, whereresidue 1 is the N-terminal methionine, would have residues 2 through Nremaining after removal of the N-terminal methionine. Alternatively, amature form arising from a precursor polypeptide or protein havingresidues 1 to N, in which an N-terminal signal sequence from residue 1to residue M is cleaved, would have the residues from residue M+1 toresidue N remaining. Further as used herein, a “mature” form of apolypeptide or protein may arise from a step of post-translationalmodification other than a proteolytic cleavage event. Such additionalprocesses include, by way of non-limiting example, glycosylation,myristoylation or phosphorylation. In general, a mature polypeptide orprotein may result from the operation of only one of these processes, ora combination of any of them.

The invention includes mutant or variant nucleic acids of SEQ ID NO:1,or a fragment thereof, any of whose bases may be changed from thecorresponding bases shown in SEQ ID NO:1, while still encoding a proteinthat maintains at least one of its BFLP0169-like activities andphysiological functions (i.e., modulating angiogenesis, neuronaldevelopment). The invention further includes the complement of thenucleic acid sequence of SEQ ID NO:1, including fragments, derivatives,analogs and homologs thereof. The invention additionally includesnucleic acids or nucleic acid fragments, or complements thereto, whosestructures include chemical modifications.

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode BFLP0169 proteins or biologically active portions thereof.Also included are nucleic acid fragments sufficient for use ashybridization probes to identify BFLP0169-encoding nucleic acids (e.g.,BFLP0169 mRNA) and fragments for use as polymerase chain reaction (PCR)primers for the amplification or mutation of BFLP0169 nucleic acidmolecules. As used herein, the term “nucleic acid molecule” is intendedto include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules(e.g., mRNA), analogs of the DNA or RNA generated using nucleotideanalogs, and derivatives, fragments and homologs thereof. The nucleicacid molecule can be single-stranded or double-stranded, but preferablyis double-stranded DNA.

“Probes” refer to nucleic acid sequences of variable length, preferablybetween at least about 10 nucleotides (nt), 100 nt, or as many as about,e.g., 6,000 nt, depending on use. Probes are used in the detection ofidentical, similar, or complementary nucleic acid sequences. Longerlength probes are usually obtained from a natural or recombinant source,are highly specific and much slower to hybridize than oligomers. Probesmay be single- or double-stranded and designed to have specificity inPCR, membrane-based hybridization technologies, or ELISA-liketechnologies.

An “isolated” nucleic acid molecule is one that is separated from othernucleic acid molecules that are present in the natural source of thenucleic acid. Examples of isolated nucleic acid molecules include, butare not limited to, recombinant DNA molecules contained in a vector,recombinant DNA molecules maintained in a heterologous host cell,partially or substantially purified nucleic acid molecules, andsynthetic DNA or RNA molecules. Preferably, an “isolated” nucleic acidis free of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated BFLP0169 nucleic acidmolecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial or culture medium when produced by recombinant techniques, orof chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, or a complementthereof, can be isolated using standard molecular biology techniques andthe sequence information provided herein. Using all or a portion of thenucleic acid sequence of SEQ ID NO:1 as a hybridization probe, BFLP0169nucleic acid sequences can be isolated using standard hybridization andcloning techniques (e.g., as described in Sambrook et al., eds.,MOLECULAR CLONING: A LABORATORY MANUAL 2^(nd) Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al.,eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NewYork, N.Y., 1993.)

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to BFLP0169 nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

As used herein, the term “oligonucleotide” refers to a series of linkednucleotide residues, which oligonucleotide has a sufficient number ofnucleotide bases to be used in a PCR reaction. A short oligonucleotidesequence may be based on, or designed from, a genomic or cDNA sequenceand is used to amplify, confirm, or reveal the presence of an identical,similar or complementary DNA or RNA in a particular cell or tissue.Oligonucleotides comprise portions of a nucleic acid sequence havingabout 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 ntin length. In one embodiment, an oligonucleotide comprising a nucleicacid molecule less than 100 nt in length would further comprise at lease6 contiguous nucleotides of SEQ ID NO:1, or a complement thereof.Oligonucleotides may be chemically synthesized and may be used asprobes.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule that is a complement of thenucleotide sequence shown in SEQ ID NO:1, or a portion of thisnucleotide sequence. A nucleic acid molecule that is complementary tothe nucleotide sequence shown in SEQ ID NO:1 is one that is sufficientlycomplementary to the nucleotide sequence shown in SEQ ID NO:1 that itcan hydrogen bond with little or no mismatches to the nucleotidesequence shown in SEQ ID NO:1, thereby forming a stable duplex.

As used herein, the term “complementary” refers to Watson-Crick orHoogsteen base pairing between nucleotide units of a nucleic acidmolecule, and the term “binding” means the physical or chemicalinteraction between two polypeptides or compounds or associatedpolypeptides or compounds or combinations thereof. Binding includesionic, non-ionic, Van der Waals, hydrophobic interactions, etc. Aphysical interaction can be either direct or indirect. Indirectinteractions may be through or due to the effects of another polypeptideor compound. Direct binding refers to interactions that do not takeplace through, or due to, the effect of another polypeptide or compound,but instead are without other substantial chemical intermediates.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, e.g., a fragmentthat can be used as a probe or primer, or a fragment encoding abiologically active portion of BFLP0169 . Fragments provided herein aredefined as sequences of at least 6 (contiguous) nucleic acids or atleast 4 (contiguous) amino acids, a length sufficient to allow forspecific hybridization in the case of nucleic acids or for specificrecognition of an epitope in the case of amino acids, respectively, andare at most some portion less than a full length sequence. Fragments maybe derived from any contiguous portion of a nucleic acid or amino acidsequence of choice. Derivatives are nucleic acid sequences or amino acidsequences formed from the native compounds either directly or bymodification or partial substitution. Analogs are nucleic acid sequencesor amino acid sequences that have a structure similar to, but notidentical to, the native compound but differs from it in respect tocertain components or side chains. Analogs may be synthetic or from adifferent evolutionary origin and may have a similar or oppositemetabolic activity compared to wild type.

Derivatives and analogs may be full length or other than full length, ifthe derivative or analog contains a modified nucleic acid or amino acid,as described below. Derivatives or analogs of the nucleic acids orproteins of the invention include, but are not limited to, moleculescomprising regions that are substantially homologous to the nucleicacids or proteins of the invention, in various embodiments, by at leastabout 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with apreferred identity of 80-99%) over a nucleic acid or amino acid sequenceof identical size or when compared to an aligned sequence in which thealignment is done by a computer homology program known in the art, orwhose encoding nucleic acid is capable of hybridizing to the complementof a sequence encoding the aforementioned proteins under stringent,moderately stringent, or low stringent conditions. An exemplary programis the Gap program (Wisconsin Sequence Analysis Package, Version 8 forUNIX, Genetics Computer Group, University Research Park, Madison, Wis.)using the default settings, which uses the algorithm of Smith andWaterman.

A “homologous nucleic acid sequence” or “homologous amino acidsequence,” or variations thereof, refer to sequences characterized by ahomology at the nucleotide level or amino acid level as discussed above.Homologous nucleotide sequences encode those sequences coding forisoforms of a BFLP0169 polypeptide. Isoforms can be expressed indifferent tissues of the same organism as a result of, for example,alternative splicing of RNA. Alternatively, isoforms can be encoded bydifferent genes. In the present invention, homologous nucleotidesequences include nucleotide sequences encoding for a BFLP0169polypeptide of species other than humans, including, but not limited to,mammals, and thus can include, e.g., mouse, rat, rabbit, dog, cat, cow,horse, and other organisms. Homologous nucleotide sequences alsoinclude, but are not limited to, naturally occurring allelic variationsand mutations of the nucleotide sequences set forth herein. A homologousnucleotide sequence does not, however, include the nucleotide sequenceencoding human BFLP0169 protein. Homologous nucleic acid sequencesinclude those nucleic acid sequences that encode conservative amino acidsubstitutions (see below) in SEQ ID NO:2, as well as a polypeptidehaving BFLP0169 activity. Biological activities of the BFLP0169 proteinsare described below. A homologous amino acid sequence does not encodethe amino acid sequence of a human BFLP0169 polypeptide.

The nucleotide sequence determined from the cloning of the humanBFLP0169 gene allows for the generation of probes and primers designedfor use in identifying and/or cloning BFLP0169 homologues in other celltypes, e.g., from other tissues, as well as BFLP0169 homologues fromother mammals. The probe/primer typically comprises a substantiallypurified oligonucleotide. The oligonucleotide typically comprises aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 ormore consecutive sense strand nucleotide sequence of SEQ ID NO:1; or ananti-sense strand nucleotide sequence of SEQ ID NO:1; or of a naturallyoccurring mutant of SEQ ID NO:1.

Probes based on the human BFLP0169 nucleotide sequence can be used todetect transcripts or genomic sequences encoding the same or homologousproteins. In various embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a BFLP0169 protein, such as by measuring a levelof a BFLP0169-encoding nucleic acid in a sample of cells from a subjecte.g., detecting BFLP0169 mRNA levels or determining whether a genomicBFLP0169 gene has been mutated or deleted.

A “polypeptide having a biologically active portion of BFLP0169 ” refersto polypeptides exhibiting activity similar, but not necessarilyidentical to, an activity of a polypeptide of the present invention,including mature forms, as measured in a particular biological assay,with or without dose dependency. A nucleic acid fragment encoding a“biologically active portion of BFLP0169” can be prepared by isolating aportion of SEQ ID NO:1 that encodes a polypeptide having a BFLP0169biological activity (biological activities of the BFLP0169 proteins aredescribed below), expressing the encoded portion of BFLP0169 protein(e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of BFLP0169.

The invention also provides polymorphic forms of BFLP0169 nucleic acidsequences as well as methods of detecting polymorphic sequences inBFLP0169 sequences The polymorphic forms include genomic sequencescorresponding to exons and/or introns associated with BFLP0169.

Individuals carrying polymorphic alleles of the invention may bedetected at either the DNA, the RNA, or the protein level using avariety of techniques that are well known in the art. The presentmethods usually employ pre-characterized polymorphisms. That is, thegenotyping location and nature of polymorphic forms present at a sitehave already been determined. The availability of this informationallows sets of probes to be designed for specific identification of theknown polymorphic forms.

The genomic DNA used for the diagnosis may be obtained from anynucleated cells of the body, such as those present in peripheral blood,urine, saliva, buccal samples, surgical specimen, and autopsy specimens.The DNA may be used directly or may be amplified enzymatically in vitrothrough use of PCR or other in vitro amplification methods such as theligase chain reaction (LCR), strand displacement amplification (SDA),self-sustained sequence replication (3SR), prior to mutation analysis.

The detection of polymorphisms in specific DNA sequences, can beaccomplished by a variety of methods including, but not limited to,restriction-fragment-length-polymorphism detection based onallele-specific restriction-endonuclease cleavage, hybridization withallele-specific oligonucleotide probes, including immobilizedoligonucleotides or oligonucleotide arrays, allele-specific PCR,mismatch-repair detection (MRD), binding of MutS protein,denaturing-gradient gel electrophoresis (DGGE),single-strand-conformation-polymorphism detection, RNAase cleavage atmismatched base-pairs, chemical or enzymatic cleavage of heteroduplexDNA, methods based on allele specific primer-extension, genetic bitanalysis (GBA), the oligonucleotide-ligation assay (OLA), theallele-specific ligation chain reaction (LCR), gap-LCR, radioactiveand/or fluorescent DNA sequencing using standard procedures well knownin the art, and peptide nucleic acid (PNA) assays.

BFLP0169 Variants

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequences shown in SEQ ID NO:1 due to the degeneracyof the genetic code. These nucleic acids thus encode the same BFLP0169protein as that encoded by the nucleotide sequence shown in SEQ ID NO:1,e.g., the polypeptide of SEQ ID NO:2. In another embodiment, an isolatednucleic acid molecule of the invention has a nucleotide sequenceencoding a protein having an amino acid sequence shown in SEQ ID NO:2.

In addition to the human BFLP0169 nucleotide sequence shown in SEQ IDNO:1, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof BFLP0169 may exist within a population (e.g., the human population).Such genetic polymorphism in the BFLP0169 gene may exist amongindividuals within a population due to natural allelic variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a BFLP0169protein, preferably a mammalian BFLP0169 protein. Such natural allelicvariations can typically result in 1-5% variance in the nucleotidesequence of the BFLP0169 gene. Any and all such nucleotide variationsand resulting amino acid polymorphisms in BFLP0169 that are the resultof natural allelic variation and that do not alter the functionalactivity of BFLP0169 are intended to be within the scope of theinvention.

Moreover, nucleic acid molecules encoding BFLP0169 proteins from otherspecies, and thus that have a nucleotide sequence that differs from thehuman sequence of SEQ ID NO:1 are intended to be within the scope of theinvention. Nucleic acid molecules corresponding to natural allelicvariants and homologues of the BFLP0169 cDNAs of the invention can beisolated based on their homology to the human BFLP0169 nucleic acidsdisclosed herein using the human cDNAs, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. For example, a soluble humanBFLP0169 cDNA can be isolated based on its homology to humanmembrane-bound BFLP0169. Likewise, a membrane-bound human BFLP0169 cDNAcan be isolated based on its homology to soluble human BFLP0169.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1. In another embodiment, the nucleicacid is at least 10, 25, 50, 100, 250, 500 or 750 nucleotides in length.In another embodiment, an isolated nucleic acid molecule of theinvention hybridizes to the coding region. As used herein, the term“hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 60% homologous to each other typically remainhybridized to each other.

Homologs (i.e., nucleic acids encoding BFLP0169 proteins derived fromspecies other than human) or other related sequences (e.g., paralogs)can be obtained by low, moderate or high stringency hybridization withall or a portion of the particular human sequence as a probe usingmethods well known in the art for nucleic acid hybridization andcloning. Thus, the present invention also includes polynucleotidescapable of hybridizing under reduced stringency conditions, morepreferably stringent conditions, and most preferably highly stringentconditions, to polynucleotides described herein. Examples of stringencyconditions are shown in the table below: highly stringent conditions arethose that are at least as stringent as, for example, conditions A-F;stringent conditions are at least as stringent as, for example,conditions G-L; and reduced stringency conditions are at least asstringent as, for example, conditions M-R.

TABLE 4 Stringency Conditions Hybrid Hybridization StringencyPolynucleotide Length Temperature Wash Temperature Condition Hybrid(bp)¹ and Buffer^(H) and Buffer^(H) A DNA:DNA ≧50 65° C.; 1 × SSC -or-65° C.; 0.3 × SSC 42° C.; 1 × SSC, 50% formamide B DNA:DNA <50 T_(B)*; 1× SSC T_(B)*; 1 × SSC 67° C.; 0.3 × SSC C DNA:RNA ≧50 67° C.; 1 × SSC-or- 45° C.; 1 × SSC, 50% formamide D DNA:RNA <50 T_(D)*; 1 × SSCT_(D)*; 1 × SSC 70° C.; 0.3 × SSC E RNA:RNA ≧50 70° C.; 1 × SSC -or- 50°C.; 1 × SSC, 50% formamide F RNA:RNA <50 T_(F)*; 1 × SSC T_(f)*; 1 × SSC65° C.; 1 × SSC G DNA:DNA ≧50 65° C.; 4 × SSC -or- 42° C.; 4 × SSC, 50%formamide H DNA:DNA <50 T_(H)*; 4 × SSC T_(H)*; 4 × SSC 67° C.; 1 × SSCI DNA:RNA ≧50 67° C.; 4 × SSC -or- 45° C.; 4 × SSC, 50% formamide JDNA:RNA <50 T_(J)*; 4 × SSC T_(J)*; 4 × SSC 67° C.; 1 × SSC K RNA:RNA≧50 70° C.; 4 × SSC -or- 50° C.; 4 × SSC, 50% formamide L RNA:RNA <50T_(L)*; 2 × SSC T_(L)*; 2 × SSC 50° C.; 2 × SSC M DNA:DNA >50 50° C.; 4× SSC -or- 40° C.; 6 × SSC, 50% formamide N DNA:DNA <50 T_(N)*; 6 × SSCT_(N)*; 6 × SSC 55° C.; 2 × SSC O DNA:RNA >50 55° C.; 4 × SSC -or- 42°C.; 6 × SSC, 50% formamide P DNA:RNA <50 T_(P)*; 6 × SSC T_(P)*; 6 × SSC60° C.; 2 × SSC Q RNA:RNA >50 60° C.; 4 × SSC -or- 45° C.; 6 × SSC, 50%formamide R RNA:RNA <50 T_(R)*; 4 × SSC T_(R)*; 4 × SSC ¹The hybridlength is that anticipated for the hybridized region(s) of thehybridizing polynucleotides. When hybridizing a polynucleotide to atarget polynucleotide of unknown sequence, the hybrid length is assumedto be that of the hybridizing polynucleotide. When polynucleotides ofknown sequence are hybridized, the hybrid length can be determined byaligning the sequences of the polynucleotides and identifying the regionor regions of optimal sequence complementarity. ^(H)SSPE (1 × SSPE is0.15 M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substitutedfor SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) in thehybridization and wash buffers; washes are performed for 15 minutesafter hybridization is complete. T_(B)*-T_(R)*: The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature (T_(m)) ofthe hybrid, where T_(m) is determined according to the followingequations. For hybrids less than 18 base pairs in length,T_(m)(° C.) =2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49base pairs in length, T_(m)(° C.) = 81.5 + 16.6(log₁₀Na⁺) + 0.41(% G +C) − (600/N), where N is the number of bases in the hybrid, and Na⁺ isthe concentration of sodium ions in the hybridization buffer (Na⁺ for 1× SSC = 0.165 M).

Preferably, each such hybridizing polynucleotide has a length that is atleast 25% (more preferably at least 50%, and most preferably at least75%) of the length of the polynucleotide of the present invention towhich it hybridizes, and has at least 60% sequence identity (morepreferably, at least 75% identity; most preferably at least 90% or 95%identity) with the polynucleotide of the present invention to which ithybridizes, where sequence identity is determined by comparing thesequences of the hybridizing polynucleotides when aligned so as tomaximize overlap and identity while minimizing sequence gaps.

A non-limiting example of stringent hybridization conditions ishybridization in a high salt buffer comprising 6×SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/mldenatured salmon sperm DNA at 65° C. This hybridization is followed byone or more washes in 0.2×SSC, 0.01% BSA at 50° C. An isolated nucleicacid molecule of the invention that hybridizes under stringentconditions to the sequence of SEQ ID NO:1 corresponds to a naturallyoccurring nucleic acid molecule. As used herein, a “naturally-occurring”nucleic acid molecule refers to an RNA or DNA molecule having anucleotide sequence that occurs in nature (e.g., encodes a naturalprotein).

In a second embodiment, a nucleic acid sequence that is hybridizable tothe nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO:1, or fragments, analogs or derivatives thereof, under conditions ofmoderate stringency is provided. A non-limiting example of moderatestringency hybridization conditions are hybridization in 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNAat 55° C., followed by one or more washes in 1×SSC, 0.1% SDS at 37° C.

In a third embodiment, a nucleic acid that is hybridizable to thenucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,or fragments, analogs or derivatives thereof, under conditions of lowstringency, is provided. A non-limiting example of low stringencyhybridization conditions are hybridization in 35% formamide, 5×SSC, 50mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C., followed by one or more washes in 2×SSC, 25 mM Tris-HCl (pH 7.4), 5mM EDTA, and 0.1% SDS at 50° C.

Conservative Mutations

In addition to naturally-occurring allelic variants of the BFLP0169sequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequence of SEQ ID NO:1, thereby leading to changes in theamino acid sequence of the encoded BFLP0169 protein, without alteringthe functional ability of the BFLP0169 protein. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in the sequence of SEQ ID NO:1. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of BFLP0169 without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. For example, altering amino acid residues that areconserved among the BFLP0169 proteins of the present invention, islikely to result in loss of activity of the BFLP0169 protein.

Another aspect of the invention pertains to nucleic acid moleculesencoding BFLP0169 proteins that contain changes in amino acid residuesthat are not essential for activity. Such BFLP0169 proteins differ inamino acid sequence from SEQ ID NO:2, yet retain biological activity. Inone embodiment, the isolated nucleic acid molecule comprises anucleotide sequence encoding a protein, wherein the protein comprises anamino acid sequence at least about 75% homologous to the amino acidsequence of SEQ ID NO:2. Preferably, the protein encoded by the nucleicacid is at least about 80% homologous to SEQ ID NO:2, more preferably atleast about 90%, 95%, 98%, and most preferably at least about 99%homologous to SEQ ID NO:2.

An isolated nucleic acid molecule encoding a BFLP0169 protein homologousto the protein of SEQ ID NO:2 can be created by introducing one or morenucleotide substitutions, additions or deletions into the nucleotidesequence of SEQ ID NO:1, such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.

Mutations can be introduced into the nucleotide sequence of SEQ ID NO:1by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue inBFLP0169 is replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a BFLP0169 coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for BFLP0169 biological activity to identify mutants thatretain activity. Following mutagenesis of SEQ ID NO:1 the encodedprotein can be expressed by any recombinant technology known in the artand the activity of the protein can be determined.

In one embodiment, a mutant BFLP0169 protein can be assayed for (1) theability to form protein:protein interactions with other BFLP0169proteins, other cell-surface proteins, or biologically active portionsthereof, (2) complex formation between a mutant BFLP0169 protein and aBFLP0169 receptor; (3) the ability of a mutant BFLP0169 protein to bindto an intracellular target protein or biologically active portionthereof; (e.g., avidin proteins); (4) the ability to bind BFLP0169protein; or (5) the ability to specifically bind an anti-BFLP0169protein antibody.

Antisense BFLP0169 Nucleic Acids

Another aspect of the invention pertains to isolated antisense nucleicacid molecules that are hybridizable to or complementary to the nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO:1, orfragments, analogs or derivatives thereof. An “antisense” nucleic acidcomprises a nucleotide sequence that is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. In specific aspects, antisense nucleic acid molecules areprovided that comprise a sequence complementary to at least about 10,25, 50, 100, 250 or 500 nucleotides or an entire BFLP0169 coding strand,or to only a portion thereof. Nucleic acid molecules encoding fragments,homologs, derivatives and analogs of a BFLP0169 protein of SEQ ID NO:2,or antisense nucleic acids complementary to a BFLP0169 nucleic acidsequence of SEQ ID NO:1 are additionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence encodingBFLP0169. The term “coding region” refers to the region of thenucleotide sequence comprising codons which are translated into aminoacid residues (e.g., the protein coding region of human BFLP0169corresponds to SEQ ID NO:2). In another embodiment, the antisensenucleic acid molecule is antisense to a “noncoding region” of the codingstrand of a nucleotide sequence encoding BFLP0169. The term “noncodingregion” refers to 5′ and 3′ sequences which flank the coding region thatare not translated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

Given the coding strand sequences encoding BFLP0169 disclosed herein(e.g., SEQ ID NO:1), antisense nucleic acids of the invention can bedesigned according to the rules of Watson and Crick or Hoogsteen basepairing. The antisense nucleic acid molecule can be complementary to theentire coding region of BFLP0169 mRNA, but more preferably is anoligonucleotide that is antisense to only a portion of the coding ornoncoding region of BFLP0169 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of BFLP0169 mRNA. An antisense oligonucleotidecan be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis or enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used.

Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a BFLP0169protein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule that binds toDNA duplexes, through specific interactions in the major groove of thedouble helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies that bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of antisense molecules,vector constructs in which the antisense nucleic acid molecule is placedunder the control of a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other. The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide.

Such modifications include, by way of nonlimiting example, modifiedbases, and nucleic acids whose sugar phosphate backbones are modified orderivatized. These modifications are carried out at least in part toenhance the chemical stability of the modified nucleic acid, such thatthey may be used, for example, as antisense binding nucleic acids intherapeutic applications in a subject.

BFLP0169 Ribozymes and PNA Moieties

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme.

Ribozymes are catalytic RNA molecules with ribonuclease activity thatare capable of cleaving a single-stranded nucleic acid, such as a mRNA,to which they have a complementary region. Thus, ribozymes (e.g.,hammerhead ribozymes) can be used to catalytically cleave BFLP0169 mRNAtranscripts to thereby inhibit translation of BFLP0169 mRNA. A ribozymehaving specificity for a BFLP0169-encoding nucleic acid can be designedbased upon the nucleotide sequence of a BFLP0169 DNA disclosed herein(i.e., SEQ ID NO:1). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the activesite is complementary to the nucleotide sequence to be cleaved in aBFLP0169-encoding mRNA. Alternatively, BFLP0169 mRNA can be used toselect a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules.

Alternatively, BFLP0169 gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of theBFLP0169 (e.g., the BFLP0169 promoter and/or enhancers) to form triplehelical structures that prevent transcription of the BFLP0169 gene intarget cells.

In various embodiments, the nucleic acids of BFLP0169 can be modified atthe base moiety, sugar moiety or phosphate backbone to improve, e.g.,the stability, hybridization, or solubility of the molecule. Forexample, the deoxyribose phosphate backbone of the nucleic acids can bemodified to generate peptide nucleic acids. As used herein, the terms“peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g.,DNA mimics, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols.

PNAs of BFLP0169 can be used in therapeutic and diagnostic applications.For example, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs ofBFLP0169 can also be used, e.g., in the analysis of single base pairmutations in a gene by, e.g., PNA directed PCR clamping; as artificialrestriction enzymes when used in combination with other enzymes, e.g.,S1 nucleases; or as probes or primers for DNA sequence andhybridization.

In another embodiment, PNAs of BFLP0169 can be modified, e.g., toenhance their stability or cellular uptake, by attaching lipophilic orother helper groups to PNA, by the formation of PNA-DNA chimeras, or bythe use of liposomes or other techniques of drug delivery known in theart. For example, PNA-DNA chimeras of BFLP0169 can be generated that maycombine the advantageous properties of PNA and DNA.

The oligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane or the blood-brainbarrier. In addition, oligonucleotides can be modified withhybridization triggered cleavage agents or intercalating agents. To thisend, the oligonucleotide may be conjugated to another molecule, e.g., apeptide, a hybridization triggered cross-linking agent, a transportagent, a hybridization-triggered cleavage agent, etc.

BFLP0169 Interfering Nucleic Acids

Also provided by the invention is an isolated double-stranded nucleicacid (DNA or RNA) that is capable of mediating specific inhibition ofBFLP0169 gene expression. In preferred embodiments, one or both strandsof the double-stranded molecule is an RNA molecule. Preferably, each RNAstrand has a length from 19-25, particularly from 19-23 nucleotides,more particularly from 20-22 nucleotides, and is capable of mediatingBFLP0169 target-specific nucleic acid modifications, particularly RNAinterference and/or DNA methylation. The double-stranded BFLP0169molecule may be double stranded or have an overhang at one or both the5′ and/or 3′ terminus. For example, the molecule may have a 3′ overhang.The length of the 3′-overhang can be, e.g., 1-6 nucleotides, 2-5nucleotides, 3-4 nucleotides, or 2 nucleotides. The length of theoverhang may be the same or different for each strand. In oneembodiment, dsRNAs are composed of two 21 nucleotide strands that arepaired such that 1, 2, or 3 nucleotide overhangs are present on bothends of the double-stranded RNA.

The RNA strands preferably have 3′-hydroxyl groups. The 5′-terminuspreferably includes a phosphate, diphosphate, triphosphate or hydroxylgroup. If desired, the 3′-overhangs may be stabilized againstdegradation. For example, they may be selected such that they consist ofpurine nucleotides, particularly adenosine or guanosine nucleotides.Alternatively, pyrimidine nucleotides may be replaced with modifiedanalogues, e.g. substitution of uridine-2 nucleotide 3′ overhangs by2′-deoxythymidine is tolerated, and does not affect the efficiency ofRNA interference. The RNA molecule may contain at least one modifiednucleotide analogue. The nucleotide analogues may be located atpositions where the target-specific activity, e.g. the RNAi mediatingactivity is not substantially affected. The modified nucleotide ispreferably present in a region at the 5′-end and/or the 3′-end of thedouble-stranded RNA molecule. In some embodiments, overhangs arestabilized by incorporating modified nucleotide analogues.

Nucleotide analogues can include sugar- or backbone-modifiedribonucleotides. Other suitable nucleotides include a non-naturallyoccurring nucleobase instead of a naturally occurring nucleobases. Forexample, analogues can include uridines or cytidines modified at the5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosinesand guanosines modified at the 8-position, e.g. 8-bromo guanosine; deazanucleotides, e.g. 7-deaza-adenosine; O- and N-alkylated nucleotides,e.g. N6-methyl adenosine are suitable. In preferred sugar-modifiedribonucleotides the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or CN, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I. In a preferredembodiment, where backbone-modified ribonucleotides are used as thephosphoester group connecting to adjacent ribonucleotides, they arereplaced by a modified group, e.g. a phosphothioate group. It should benoted that the above modifications may be combined.

The BFLP0169 interfering RNA molecule can be a naturally isolated RNAmolecule or can a synthetic RNA molecule. Preferably, the BFLP0169interfering RNA molecule is substantially free from contaminantsoccurring in cell extracts, e.g. from Drosophila embryos. Further, theBFLP0169 interfering RNA molecule is preferably substantially free fromany non-target-specific contaminants, particularly non-target-specificRNA molecules e.g. from contaminants occurring in cell extracts.

Isolated double-stranded BFLP0169 interfering molecules can be used formediating BFLP0169 target-specific nucleic acid modifications,particularly RNAi, in mammalian cells, particularly in human cells.

The sequence of the double-stranded BFLP0169 interfering molecule of thepresent invention is of sufficient identity to a nucleic acid BFLP0169target molecule in order to effect target-specific interference ofBFLP0169 gene expression and/or DNA methylation. Preferably, thesequence has an identity of at least 50%, particularly of at least 70%to the desired target molecule in the double-stranded portion of the RNAmolecule. More preferably, the identity is at least 85% and mostpreferably 100% in the double-stranded portion of the RNA molecule. Theidentity of a BFLP0169 double-stranded interfering RNA molecule to apredetermined nucleic acid target molecule, e.g. an BFLP0169 mRNA targetmolecule with the sequence shown in SEQ ID NO:1, may be determined usingthe equation: I=(n/L)×100, wherein I is the identity in percent, n isthe number of identical nucleotides in the double-stranded portion ofthe ds RNA and the target and L is the length of the sequence overlap ofthe double-stranded portion of the dsRNA and the target.

Alternatively, the identity of the double-stranded RNA molecule relativeto the target sequence may also be defined including the 3′ overhang,particularly an overhang having a length from 1-3 nucleotides. In thiscase the sequence identity is preferably at least 50%, more preferablyat least 70% and most preferably at least 85% to the target sequence.For example, the nucleotides from the 3′ overhang and up to 2nucleotides from the 5′ and/or 3′ terminus of the double strand may bemodified without significant loss of activity.

A double-stranded BFLP0169 RNA molecule may be prepared by a method thatincludes synthesizing two RNA strands each having a length from 19-25,e.g. from 19-23 nucleotides, wherein said RNA strands are capable offorming a double-stranded RNA molecule, wherein preferably at least onestrand has a 3′-overhang from 1-5 nucleotides, and (b) combining thesynthesized RNA strands under conditions, wherein a a double-strandedRNA molecule is formed. The double-stranded RNA molecule is capable ofmediating target-specific nucleic acid modifications, particularly RNAinterference and/or DNA methylation.

Methods of synthesizing RNA molecules are known in the art. Thesingle-stranded RNAs can also be prepared by enzymatic transcriptionfrom synthetic DNA templates or from DNA plasmids isolated fromrecombinant bacteria. Typically, phage RNA polymerases are used such asT7, T3 or SP6 RNA polymerase.

A further aspect of the present invention relates to a method ofmediating BFLP0169-specific nucleic acid modifications, particularly RNAinterference and/or DNA methylation in a cell or an organism bycontacting the cell or organism with the double-stranded RNA molecule ofthe invention under conditions wherein target-specific nucleic acidmodifications may occur and mediating a target-specific nucleic acidmodification effected by the double-stranded RNA towards a BFLP0169target nucleic acid.

BFLP0169 Polypeptides

A BFLP0169 polypeptide of the invention includes the BFLP0169-likeprotein whose sequence is provided in SEQ ID NO:2. The invention alsoincludes a mutant or variant form of the disclosed BFLP0169 polypeptide,or of any of the fragments of the herein disclosed BFLP0169 polypeptidesequences.

Thus, a BFLP0169 polypeptide includes one in which any residues may bechanged from the corresponding residue shown in SEQ ID NO:2 while stillencoding a protein that maintains its BFLP0169-like activities andphysiological functions, or a functional fragment thereof. In someembodiments, up to 20% or more of the residues may be so changed in themutant or variant protein. In some embodiments, the BFLP0169 polypeptideaccording to the invention is a mature polypeptide.

Rapamycin Binding Domains

To identify regions of a BFLP0169 polypeptide sequence (e.g., apolypeptide including all or a portion of SEQ ID NO:2) containingrapamycin binding domains, the entire coding sequence, or a fragment ofa BFLP0169 polypeptide sequence, is tested for its ability to bindrapamycin. Any technique known in the art for determining binding of apolypeptide to a small molecule can be used. For example, rapamycin canbe labeled (i.e., with a non-radioactive label or with a radiolabel(e.g.,¹⁴C, ³²P, ³H, or ¹²⁵I), and mixed with a polypeptide containingsome or all of a BFLP0169 polypeptide sequence. The polypeptideoptionally includes a moiety that facilitates detection, e.g., thepolypeptide can be a fusion polypeptide that includes a BFLP0169sequence and a non-BFLP0169 polypeptide sequence.

A reagent specific for the polypeptide containing the BFLP0169polypeptide sequence (e.g., an antibody specific for BFLP0169 or a probespecific for the non-BFLP0169 polypeptide in the case of a fusionpolypeptide) is added to the mixture. Complexes that bind to the reagentare isolated, and the presence of label, which reveals the presence ofrapamycin, is determined.

In general, a BFLP0169-like variant that preserves BFLP0169-likefunction includes any variant in which residues at a particular positionin the sequence have been substituted by other amino acids, and furtherinclude the possibility of inserting an additional residue or residuesbetween two residues of the parent protein as well as the possibility ofdeleting one or more residues from the parent sequence. Any amino acidsubstitution, insertion, or deletion is encompassed by the invention. Infavorable circumstances, the substitution is a conservative substitutionas defined above.

One aspect of the invention pertains to isolated BFLP0169 proteins, andbiologically active portions thereof, or derivatives, fragments, analogsor homologs thereof. Fragments can comprise contigous stretches of SEQID NO:2, or interspersed segments of SEQ ID NO:2. Also provided arepolypeptide fragments suitable for use as immunogens to raiseanti-BFLP0169 antibodies. In one embodiment, native BFLP0169 proteinscan be isolated from cells or tissue sources by an appropriatepurification scheme using standard protein purification techniques. Inanother embodiment, BFLP0169 proteins are produced by recombinant DNAtechniques. Alternative to recombinant expression, a BFLP0169 protein orpolypeptide can be synthesized chemically using standard peptidesynthesis techniques.

A “purified” protein or biologically active portion thereof issubstantially free of cellular material or other contaminating proteinsfrom the cell or tissue source from which the BFLP0169 protein isderived, or substantially free from chemical precursors or otherchemicals when chemically synthesized. The language “substantially freeof cellular material” includes preparations of BFLP0169 protein in whichthe protein is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. In one embodiment, thelanguage “substantially free of cellular material” includes preparationsof BFLP0169 protein having less than about 30% (by dry weight) ofnon-BFLP0169 protein (also referred to herein as a “contaminatingprotein”), more preferably less than about 20% of non-BFLP0169 protein,still more preferably less than about 10% of non-BFLP0169 protein, andmost preferably less than about 5% non-BFLP0169 protein. When theBFLP0169 protein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of BFLP0 169 protein in which theprotein is separated from chemical precursors or other chemicals thatare involved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of BFLP0169 protein having less than about 30% (bydry weight) of chemical precursors or non-BFLP0 169 chemicals, morepreferably less than about 20% chemical precursors or non-BFLP0169chemicals, still more preferably less than about 10% chemical precursorsor non-BFLP0169 chemicals, and most preferably less than about 5%chemical precursors or non-BFLP0169 chemicals.

Biologically active portions of a BFLP0169 protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the BFLP0169 protein, e.g., the aminoacid sequence shown in SEQ ID NO:2 that include fewer amino acids thanthe full length BFLP0169 proteins, and exhibit at least one activity ofa BFLP0169 protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the BFLP0169 protein. Abiologically active portion of a BFLP0169 protein can be a polypeptidewhich is, for example, 10, 25, 50, 100 or more amino acids in length.

A biologically active portion of a BFLP0169 protein of the presentinvention may contain at least one of the above-identified domainsconserved between the BFLP0169 proteins. Moreover, other biologicallyactive portions, in which other regions of the protein are deleted, canbe prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native BFLP0169 protein.

In an embodiment, the BFLP0169 protein has an amino acid sequence shownin SEQ ID NO:2. In other embodiments, the BFLP0169 protein issubstantially homologous to SEQ ID NO:2 and retains the functionalactivity of the protein of SEQ ID NO:2, yet differs in amino acidsequence due to natural allelic variation or mutagenesis, as describedin detail below. Accordingly, in another embodiment, the BFLP0169protein is a protein that comprises an amino acid sequence at leastabout 45% homologous to the amino acid sequence of SEQ ID NO:2 andretains the functional activity of the BFLP0169 proteins of SEQ ID NO:2.

Determining Homology Between Two or More Sequences

To determine the percent homology of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in either of the sequences beingcompared for optimal alignment between the sequences). The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules arehomologous at that position (i.e., as used herein amino acid or nucleicacid “homology” is equivalent to amino acid or nucleic acid “identity”).

The nucleic acid sequence homology may be determined as the degree ofidentity between two sequences. The homology may be determined usingcomputer programs known in the art, such as GAP software provided in theGCG program package. Using GCG GAP software with the following settingsfor nucleic acid sequence comparison: GAP creation penalty of 5.0 andGAP extension penalty of 0.3, the coding region of the analogous nucleicacid sequences referred to above exhibits a degree of identitypreferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, withthe CDS (encoding) part of the DNA sequence shown in SEQ ID NO:1.

The term “sequence identity” refers to the degree to which twopolynucleotide or polypeptide sequences are identical on aresidue-by-residue basis over a particular region of comparison. Theterm “percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over that region of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I, in the case of nucleic acids) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the region ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The term “substantialidentity” as used herein denotes a characteristic of a polynucleotidesequence, wherein the polynucleotide comprises a sequence that has atleast 80 percent sequence identity, preferably at least 85 percentidentity and often 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison region. The term “percentage of positive residues” iscalculated by comparing two optimally aligned sequences over that regionof comparison, determining the number of positions at which theidentical and conservative amino acid substitutions, as defined above,occur in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the region of comparison (i.e., the window size), andmultiplying the result by 100 to yield the percentage of positiveresidues.

Chimeric and Fusion Proteins

The invention also provides BFLP0169 chimeric or fusion proteins. Asused herein, a BFLP0169 “chimeric protein” or “fusion protein” comprisesa BFLP0169 polypeptide operatively linked to a non-BFLP0169 polypeptide.A “BFLP0169 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to BFLP0169, whereas a “non-BFLP0169 polypeptide”refers to a polypeptide having an amino acid sequence corresponding to aprotein that is not substantially homologous to the BFLP0169 protein,e.g., a protein that is different from the BFLP0169 protein and that isderived from the same or a different organism. Within a BFLP0169 fusionprotein the BFLP0169 polypeptide can correspond to all or a portion of aBFLP0169 protein. An example of a BFLP0169 fusion polypeptide is onethat includes amino acids 21-230 of SEQ ID NO:2 (e.g., a polypeptidethat includes amino acids 1-246 or amino acids 21-246 of SEQ ID NO:2).In one embodiment, a BFLP0169 fusion protein comprises at least onebiologically active portion of a BFLP0169 protein. In anotherembodiment, a BFLP0169 fusion protein comprises at least twobiologically active portions of a BFLP0169 protein. Within the fusionprotein, the term “operatively linked” is intended to indicate that theBFLP0169 polypeptide and the non-BFLP0169 polypeptide are fused in-frameto each other. The non-BFLP0169 polypeptide can be fused to theN-terminus or C-terminus of the BFLP0169 polypeptide.

For example, in one embodiment a BFLP0169 fusion protein comprises aBFLP0169 polypeptide operably linked to either an extracellular domainof a second protein, i.e., non-BFLP0169 protein, or to the transmembraneand intracellular domain of a second protein, i.e., non-BFLP0169protein. Such fusion proteins can be further utilized in screeningassays for compounds that modulate BFLP0169 activity (such assays aredescribed in detail below).

In another embodiment, the fusion protein is a GST-BFLP0169 fusionprotein in which the BFLP0169 sequences are fused to the C-terminus ofthe GST (i.e., glutathione S-transferase) sequences. Such fusionproteins can facilitate the purification of recombinant BFLP0169.

In another embodiment, the fusion protein is a BFLP0169-immunoglobulinfusion protein in which the BFLP0169 sequences comprising one or moredomains are fused to sequences derived from a member of theimmunoglobulin protein family.

Inhibition of the BFLP0169 ligand/BFLP0169 interaction can be usedtherapeutically for both the treatment of proliferative anddifferentiative disorders, e.g., cancer, modulating (e.g., promoting orinhibiting) cell survival as well as immunomodulatory disorders,autoimmunity, transplantation, and inflammation by alteration ofcyotokine and chemokine cascade mechanisms. Moreover, theBFLP0169-immunoglobulin fusion proteins of the invention can be used asimmunogens to produce anti-BFLP0169 antibodies in a subject, to purifyBFLP0169 ligands, and in screening assays to identify molecules thatinhibit the interaction of BFLP0169 with a BFLP0169 ligand.

A BFLP0169 chimeric or fusion protein of the invention can be producedby standard recombinant DNA techniques. For example, DNA fragmentscoding for the different polypeptide sequences are ligated togetherin-frame in accordance with conventional techniques, e.g., by employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence.Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). ABFLP0169-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the BFLP0169protein.

If desired, libraries of fragments of the BFLP0169 protein codingsequence can be used to generate a variegated population of BFLP0169fragments for screening and subsequent selection of variants of aBFLP0169 protein.

BFLP0169 Antibodies

Also included in the invention are antibodies to BFLP0169 proteins, orfragments of BFLP0169 proteins. The term “antibody” as used hereinrefers to immunoglobulin molecules and immunologically active portionsof immunoglobulin (Ig) molecules, i.e., molecules that contain anantigen binding site that specifically binds (immunoreacts with) anantigen. Such antibodies include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, F_(ab), F_(ab′) and F_((ab′)2)fragments, and an Fab expression library. In general, an antibodymolecule obtained from humans relates to any of the classes IgG, IgM,IgA, IgE and IgD, which differ from one another by the nature of theheavy chain present in the molecule. Certain classes have subclasses aswell, such as IgG₁, IgG₂, and others. Furthermore, in humans, the lightchain may be a kappa chain or a lambda chain. Reference herein toantibodies includes a reference to all such classes, subclasses andtypes of human antibody species.

An isolated BFLP0169-related protein of the invention may be intended toserve as an antigen, or a portion or fragment thereof, and additionallycan be used as an immunogen to generate antibodies thatimmunospecifically bind the antigen, using standard techniques forpolyclonal and monoclonal antibody preparation. The full-length proteincan be used or, alternatively, the invention provides antigenic peptidefragments of the antigen for use as immunogens. An antigenic peptidefragment comprises at least 6 amino acid residues of the amino acidsequence of the full length protein, such as an amino acid sequenceshown in SEQ ID NO:2, and encompasses an epitope thereof such that anantibody raised against the peptide forms a specific immune complex withthe full length protein or with any fragment that contains the epitope.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, or at least 15 amino acid residues, or at least 20 amino acidresidues, or at least 30 amino acid residues. Preferred epitopesencompassed by the antigenic peptide are regions of the protein that arelocated on its surface; commonly these are hydrophilic regions.

In certain embodiments of the invention, at least one epitopeencompassed by the antigenic peptide is a region of BFLP0169-relatedprotein that is located on the surface of the protein, e.g., ahydrophilic region. A hydrophobicity analysis of the humanBFLP0169-related protein sequence will indicate which regions of aBFLP0169-related protein are particularly hydrophilic and, therefore,are likely to encode surface residues useful for targeting antibodyproduction. As a means for targeting antibody production, hydropathyplots showing regions of hydrophilicity and hydrophobicity may begenerated by any method well known in the art, including, for example,the Kyte Doolittle or the Hopp Woods methods, either with or withoutFourier transformation.

The novel nucleic acid encoding the BFLP0169 protein of the invention,or fragments thereof, may further be useful in diagnostic applications,wherein the presence or amount of the nucleic acid or the protein are tobe assessed. These materials are further useful in the generation ofantibodies that bind immunospecifically to the novel substances of theinvention for use in therapeutic or diagnostic methods. The disclosedBFLP0169 protein hasmultiple hydrophilic regions, each of which can beused as an immunogen. In one embodiment, a contemplated BFLP0169 epitopeis from about amino acids 20 to 90. In another embodiment, a BFLP0169epitope is from about amino acids 100 to 130. In additional embodiments,BFLP0169 epitopes are from about amino acids 140 to 220, from aboutamino acids 240 to 250, from about amino acids 280 to 290, from aboutamino acids 330 to 340, from about amino acids 370 to 380, from aboutamino acids 400 to 410, from about amino acids 450 to 520, from aboutamino acids 530 to 540, from about amino acids 640 to 650, from aboutamino acids 720 to 730, from about amino acids 800 to 820, from aboutamino acids 850 to 855, from about amino acids 900 to 910, from aboutamino acids 920 to 930, from about amino acids 940 to 950, from aboutamino acids 970 to 990, from about amino acids 1000 to 1030, from aboutamino acids 1060 to 1080, from about amino acids 1100 to 1110, fromabout amino acids 1170 to 1180, from about amino acids 1190 to 1210,from about amino acids 1250 to 1280, from about amino acids 1310 to1320, from about amino acids 1350 to 1370, from about amino acids 1400to 1420, from about amino acids 1430 to 1440, from about amino acids1500 to 1560, from about amino acids 1600 to 1610, from about aminoacids 1650 to 1690, from about amino acids 1700 to 1710, and from aboutamino acids 1720 to 1730.

Antibodies that are specific for one or more domains within an antigenicprotein, or derivatives, fragments, analogs or homologs thereof, arealso provided herein.

A protein of the invention, or a derivative, fragment, analog, homologor ortholog thereof, may be utilized as an immunogen in the generationof antibodies that immunospecifically bind these protein components.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against a protein of theinvention, or against derivatives, fragments, analogs homologs ororthologs thereof . The term “monoclonal antibody” (MAb) or “monoclonalantibody composition”, as used herein, refers to a population ofantibody molecules that contain only one molecular species of antibodymolecule consisting of a unique light chain gene product and a uniqueheavy chain gene product.

The antibodies directed against the protein antigens of the inventioncan further comprise humanized antibodies or human antibodies. Thehumanized forms of antibodies include chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) that areprincipally comprised of the sequence of a human immunoglobulin, andcontain minimal sequence derived from a non-human immunoglobulin.

The antibodies can also be human antibodies, e.g., antibody molecules inwhich essentially the entire sequences of both the light chain and theheavy chain, including the CDRs, arise from human genes. Humanmonoclonal antibodies can be prepared by the trioma technique; the humanB-cell hybridoma technique and the EBV hybridoma technique.

Human antibodies can also be produced using phage display libraries, orby introducing human immunoglobulin loci into transgenic animals, e.g.,mice in which the endogenous immunoglobulin genes have been partially orcompletely inactivated. Human antibodies may additionally be producedusing transgenic nonhuman animals that are modified so as to producefully human antibodies rather than the animal's endogenous antibodies inresponse to challenge by an antigen.

The invention also provides single-chain antibodies specific to anantigenic protein of the invention. In addition, methods can be adaptedfor the construction of F_(ab) expression libraries to allow rapid andeffective identification of monoclonal F_(ab) fragments with the desiredspecificity for a protein or derivatives, fragments, analogs or homologsthereof. Antibody fragments that contain the idiotypes to a proteinantigen may be produced by techniques known in the art including, butnot limited to: (i) an F_((ab)2) fragment produced by pepsin digestionof an antibody molecule; (ii) an F_(ab) fragment generated by reducingthe disulfide bridges of an F_((ab′)2) fragment; (iii) an F_(ab)fragment generated by the treatment of the antibody molecule with papainand a reducing agent and (iv) F_(v) fragments.

Also provided by the invention are bispecific antibodies. Bispecificantibodies are monoclonal, preferably human or humanized, antibodiesthat have binding specificities for at least two different antigens. Oneof the binding specificities is for an antigenic protein of theinvention. The second binding target is any other antigen, andadvantageously is a cell-surface protein or receptor or receptorsubunit.

If desired, antibody variable domains with the desired bindingspecificities (antibody-antigen combining sites) can be fused toimmunoglobulin constant domain sequences. The fusion preferably is withan immunoglobulin heavy-chain constant domain, comprising at least partof the hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions.

Bispecific antibodies can be provided as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies).

Also within the invention are antibodies with more than two valencies(such as trispecific antibodies).

Exemplary bispecific antibodies bind to two different epitopes, at leastone of which originates in the protein antigen of the invention.

The invention also includes heteroconjugate antibodies, which includetwo covalently joined antibodies.

The antibody of the invention can be modified to alter (e.g., enhance ordiminish) its function. For example, cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchanging disulfidebond formation in this region. The invention also includesimmunoconjugates that include an antibody conjugated to a cytotoxicagent such as a chemotherapeutic agent, toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

The antibody can be conjugated to a “receptor” (such streptavidin) forutilization in tumor pretargeting wherein the antibody-receptorconjugate is administered to the patient, followed by removal of unboundconjugate from the circulation using a clearing agent and thenadministration of a “ligand” (e.g., avidin) that is in turn conjugatedto a cytotoxic agent.

BFLP0169 Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a BFLP0169protein, or derivatives, fragments, analogs or homologs thereof. As usedherein, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genomicsequence into which they have integreated. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively-linked. Such vectors are referred to herein as “expressionvectors”. “Plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

Within a recombinant expression vector, “operably-linked” is intended tomean that the nucleotide sequence of interest is linked to theregulatory sequence(s) in a manner that allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell).The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Regulatory sequences include those that direct constitutiveexpression of a nucleotide sequence in many types of host cell and thosethat direct expression of the nucleotide sequence only in certain hostcells (e.g., tissue-specific regulatory sequences). The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein (e.g., BFLP0169 proteins,mutant forms of BFLP0169 proteins, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of BFLP0169 proteins in prokaryotic or eukaryotic cells. Forexample, BFLP0169 proteins can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors)yeast cells or mammalian cells. Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 and pMT2PC. When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, adenovirus 2, cytomegalovirus, andsimian virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific),lymphoid-specific promoters, in particular promoters of T cell receptorsand immunoglobulins, neuron-specific promoters (e.g., the neurofilamentpromoter), pancreas-specific promoters, and mammary gland-specificpromoters (e.g., milk whey promoter). Developmentally-regulatedpromoters are also encompassed, e.g., the murine hox promoters and theα-fetoprotein promoter.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively-linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to BFLP0169 mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen that direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen that directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,BFLP0169 protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as human, Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells are known tothose skilled in the art.

A gene that encodes a selectable marker (e.g., resistance toantibiotics) is generally introduced into the host cells along with thegene of interest. Various selectable markers include those that conferresistance to drugs, such as G418, hygromycin and methotrexate. Anucleic acid encoding a selectable marker can be introduced into a hostcell on the same vector as that encoding BFLP0169 or can be introducedon a separate vector. Cells stably transfected with the introducednucleic acid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) BFLP0169protein. Accordingly, the invention further provides methods forproducing BFLP0169 protein using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of invention(into which a recombinant expression vector encoding BFLP0169 proteinhas been introduced) in a suitable medium such that BFLP0169 protein isproduced. In another embodiment, the method further comprises isolatingBFLP0169 protein from the medium or the host cell.

Transgenic BFLP0169 Animals

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichBFLP0169 protein-coding sequences have been introduced. Such host cellscan then be used to create non-human transgenic animals in whichexogenous BFLP0169 sequences have been introduced into their genome orhomologous recombinant animals in which endogenous BFLP0169 sequenceshave been altered. Such animals are useful for studying the functionand/or activity of BFLP0169 protein and for identifying and/orevaluating modulators of BFLP0169 protein activity. As used herein, a“transgenic animal” is a non-human animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include non-human primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA that is integrated intothe genome of a cell from which a transgenic animal develops and thatremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous BFLP0169 gene has beenaltered by homologous recombination between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducingBFLP0169-encoding nucleic acid into the male pronuclei of a fertilizedoocyte (e.g., by microinjection, retroviral infection) and allowing theoocyte to develop in a pseudopregnant female foster animal. Sequencesincluding SEQ ID NO:1 can be introduced as a transgene into the genomeof a non-human animal. Alternatively, a non-human homologue of the humanBFLP0169 gene, such as a mouse BFLP0169 gene, can be isolated based onhybridization to the human BFLP0169 cDNA (described further supra) andused as a transgene. Intronic sequences and polyadenylation signals canalso be included in the transgene to increase the efficiency ofexpression of the transgene. A tissue-specific regulatory sequence(s)can be operably-linked to the BFLP0169 transgene to direct expression ofBFLP0169 protein to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art. Similar methods areused for production of other transgenic animals. A transgenic founderanimal can be identified based upon the presence of the BFLP0169transgene in its genome and/or expression of BFLP0169 mRNA in tissues orcells of the animals. A transgenic founder animal can then be used tobreed additional animals carrying the transgene. Moreover, transgenicanimals carrying a transgene-encoding BFLP0169 protein can further bebred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a BFLP0169 gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the BFLP0169 gene. The BFLP0169 gene can be ahuman gene (e.g., the DNA of SEQ ID NO:1), but more preferably, is anon-human homologue of a human BFLP0169 gene. For example, a mousehomologue of human BFLP0169 gene of SEQ ID NO:1 can be used to constructa homologous recombination vector suitable for altering an endogenousBFLP0169 gene in the mouse genome. In one embodiment, the vector isdesigned such that, upon homologous recombination, the endogenousBFLP0169 gene is functionally disrupted (i.e., no longer encodes afunctional protein; also referred to as a “knock out” vector).

Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous BFLP0169 gene is mutated or otherwisealtered but still encodes functional protein (e.g., the upstreamregulatory region can be altered to thereby alter the expression of theendogenous BFLP0169 protein). In the homologous recombination vector,the altered portion of the BFLP0169 gene is flanked at its 5′- and3′-termini by additional nucleic acid of the BFLP0169 gene to allow forhomologous recombination to occur between the exogenous BFLP0169 genecarried by the vector and an endogenous BFLP0169 gene in an embryonicstem cell. The additional flanking BFLP0169 nucleic acid is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′- and 3′-termini) are included in the vector. The vector is thenintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced BFLP0169 gene hashomologously-recombined with the endogenous BFLP0169 gene are selected.

The selected cells are then injected into a blastocyst of an animal(e.g., a mouse) to form aggregation chimeras. A chimeric embryo can thenbe implanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously-recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously-recombined DNA by germlinetransmission of the transgene.

In another embodiment, transgenic non-humans animals can be producedthat contain selected systems that allow for regulated expression of thetransgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system. Another example of a recombinase system is the FLPrecombinase system of Saccharomyces cerevisiae. If a cre/loxPrecombinase system is used to regulate expression of the transgene,animals containing transgenes encoding both the Cre recombinase and aselected protein are required. Such animals can-be provided through theconstruction of “double” transgenic animals, e.g., by mating twotransgenic animals, one containing a transgene encoding a selectedprotein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in the art. In brief, a cell(e.g., a somatic cell) from the transgenic animal can be isolated andinduced to exit the growth cycle and enter G₀ phase. The quiescent cellcan then be fused, e.g., through the use of electrical pulses, to anenucleated oocyte from an animal of the same species from which thequiescent cell is isolated. The reconstructed oocyte is then culturedsuch that it develops to morula or blastocyte and then transferred topseudopregnant female foster animal. The offspring borne of this femalefoster animal will be a clone of the animal from which the cell (e.g.,the somatic cell) is isolated.

Methods of Detecting BFLP0169 Nucleic Acids and Diagnosing LupusNephritis

Reagents that detect BFLP0169 nucleic acids and/or polypeptides can beused to detect levels of BFLP0169 RNA and/or proteins sequences in asample. Because elevated levels of BFLP0169 RNA are found in animalswith lupus nephritis, detection of enhanced levels of BFLP0169 RNAand/or BFLP0169 polypeptides indicates the presence or predisposition tolupus in the subject. In addition, lowered levels of BFLP0169 RNA intreated lupus subjects as compared to untreated lupus indicates a returnto a non-lupus state. Thus, the efficacy of lupus treatment can bemonitored by comparing BFLP0169 RNA or protein levels in a sample from atreated population to samples in a diseased but untreated sample, (or asample from an individual that has been treated for a shorter period oftime).

Levels of BFLP0169 RNA can be assessed by comparing levels in a testcell population, from a subject whose lupus status is unknown, to levelsin a reference cell population whose lupus status is known. Thus, thetest cell population will typically include at least one cell that iscapable of expressing a BFLP0169 gene. By “capable of expressing” ismeant that the gene is present in an intact form in the cell and can beexpressed. Expression of the BFLP0169 sequence is then detected, ifpresent, and, preferably, measured using methods known in the art. Forexample, the BFLP0169 sequences disclosed herein can be used toconstruct probes for detecting BFLP0169 RNA sequences in, e.g., northernblot hybridization analyses or methods which specifically, and,preferably, quantitatively amplify BFLP0169 specific nucleic acidsequences. Alternatively, the sequences can be used to construct primersfor specifically amplifying the BFLP0169 sequences in, e.g.,amplification-based detection methods such as reverse-transcriptionbased polymerase chain reaction.

BFLP0169 expression can be also measured at the protein level, i.e., bymeasuring the levels of BFLP0169 polypeptides. Such methods are wellknown in the art and include, e.g., immunoassays based on antibodies toproteins encoded by the genes.

Expression of sequences in test and control populations of cells can becompared using any art-recognized method for comparing expression ofnucleic acid sequences. Whether or not comparison of the gene expressionprofile in the test cell population to the reference cell populationreveals the presence, or degree, of the measured parameter depends onthe composition of the reference cell population. For example, if thereference cell population is composed of cells from a lupus freesubject, a similar gene expression level in the test cell population anda reference cell population indicates the test cell population is from alupus free subject. Conversely, if the reference cell population is madeup of cells from a diseased subject, a similar gene expression profilebetween the test cell population and the reference cell populationindicates the test cell population is from a subject with lupus.

In various embodiments, a BFLP0169 sequence in a test cell population isconsidered comparable in expression level to the expression level of theADIPO sequence in the reference cell population if its expression levelvaries within a factor of 2.0, 1.5, or 1.0 fold to the level of theBFLP0169 transcript in the reference cell population. In variousembodiments, a BFLP0169 sequence in a test cell population can beconsidered altered in levels of expression if its expression levelvaries from the reference cell population by more than 1.0, 1.5, 2.0 ormore fold from the expression level of the corresponding BFLP0169sequence in the reference cell population.

If desired, comparison of differentially expressed sequences between atest cell population and a reference cell population can be done withrespect to a control nucleic acid whose expression is independent of theparameter or condition being measured. Expression levels of the controlnucleic acid in the test and reference nucleic acid can be used tonormalize signal levels in the compared populations. Suitable controlnucleic acids can readily be determined by one of ordinary skill in theart.

In some embodiments, the test cell population is compared to multiplereference cell populations. Each of the multiple reference populationsmay differ in the known parameter. Thus, a test cell population may becompared to a first reference cell population from a subject known tohave lupus, as well as a second reference population known to not havelupus.

The test cell population that is exposed can be any number of cells, ie., one or more cells, and can be provided in vitro, in vivo, or exvivo.

Preferably, cells in the reference cell population are derived from atissue type as similar as possible to test cell, e.g., renal tissue. Insome embodiments, the control cell is derived from the same subject asthe test cell. In other embodiments, the reference cell population isderived from a plurality of cells from multiple subjects. For example,the reference cell population can be a database of expression patternsfrom previously tested cells.

The subject is preferably a mammal. The mammal can be, e.g., a human,non-human primate, mouse, rat, dog, cat, horse, or cow.

Pharmaceutical Compositions

The BFLP0169 nucleic acid molecules, BFLP0169 proteins, andanti-BFLP0169 antibodies (also referred to herein as “active compounds”)of the invention, and derivatives, fragments, analogs and homologsthereof, can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the nucleicacid molecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein, “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion.

Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides,copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Screening and Detection Methods

The isolated nucleic acid molecules of the invention can be used toexpress BFLP0169 protein (e.g., via a recombinant expression vector in ahost cell in gene therapy applications), to detect BFLP0169 mRNA (e.g.,in a biological sample) or a genetic lesion in a BFLP0169 gene, and tomodulate BFLP0169 activity, as described further, below. In addition,the BFLP0169 proteins can be used to screen drugs or compounds thatmodulate the BFLP0169 protein activity or expression as well as to treatdisorders characterized by insufficient or excessive production ofBFLP0169 protein or production of BFLP0169 protein forms that havedecreased or aberrant activity compared to BFLP0169 wild-type protein.In addition, the anti-BFLP0169 antibodies of the invention can be usedto detect and isolate BFLP0169 proteins and modulate BFLP0169 activity.For example, BFLP0169 activity includes T-cell or NK cell growth anddifferentiation, antibody production, and tumor growth.

The invention further pertains to novel agents identified by thescreening assays described herein and uses thereof for treatments asdescribed, supra.

Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)that bind to BFLP0169 proteins or have a stimulatory or inhibitoryeffect on, e.g., BFLP0169 protein expression or BFLP0169 proteinactivity. The invention also includes compounds identified in thescreening assays described herein.

In one embodiment, the screening assays are used to identify therapeuticagents for treating autoimmune diseases. The autoimmune disease can be,e.g., lupus, including lupus nephtitis.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of themembrane-bound form of a BFLP0169 protein or polypeptide orbiologically-active portion thereof. The test compounds of the inventioncan be obtained using any of the numerous approaches in combinatoriallibrary methods known in the art, including: biological libraries;spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the “one-beadone-compound” library method; and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds.

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., rapamycin, nucleicacids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids orother organic or inorganic molecules. Libraries of chemical and/orbiological mixtures, such as fungal, bacterial, or algal extracts, areknown in the art and can be screened with any of the assays of theinvention. The libraries of compounds may be presented in solution, oron beads, on chips, bacteria, spores, plasmids or on phage

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a membrane-bound form of BFLP0169 protein, or abiologically-active portion thereof, on the cell surface is contactedwith a test compound and the ability of the test compound to bind to aBFLP0169 protein determined. The cell, for example, can be of mammalianorigin or a yeast cell. Determining the ability of the test compound tobind to the BFLP0169 protein can be accomplished, for example, bycoupling the test compound with a radioisotope or enzymatic label suchthat binding of the test compound to the BFLP0169 protein orbiologically-active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically-labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product. Inone embodiment, the assay comprises contacting a cell which expresses amembrane-bound form of BFLP0169 protein, or a biologically-activeportion thereof, on the cell surface with a known compound which bindsBFLP0169 to form an assay mixture, contacting the assay mixture with atest compound, and determining the ability of the test compound tointeract with a BFLP0169 protein, wherein determining the ability of thetest compound to interact with a BFLP0169 protein comprises determiningthe ability of the test compound to preferentially bind to BFLP0169protein or a biologically-active portion thereof as compared to theknown compound.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of BFLP0169 protein,or a biologically-active portion thereof, on the cell surface with atest compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the BFLP0169protein or biologically-active portion thereof. Determining the abilityof the test compound to modulate the activity of BFLP0169 or abiologically-active portion thereof can be accomplished, for example, bydetermining the ability of the BFLP0169 protein to bind to or interactwith a BFLP0169 target molecule. As used herein, a “target molecule” isa molecule with which a BFLP0169 protein binds or interacts in nature,for example, a molecule on the surface of a cell which expresses aBFLP0169 interacting protein, a molecule on the surface of a secondcell, a molecule in the extracellular milieu, a molecule associated withthe internal surface of a cell membrane or a cytoplasmic molecule. ABFLP0169 target molecule can be a non-BFLP0169 molecule or a BFLP0169protein or polypeptide of the invention In one embodiment, a BFLP0169target molecule is a component of a signal transduction pathway thatfacilitates transduction of an extracellular signal (e.g. a signalgenerated by binding of a compound to a membrane-bound BFLP0169molecule) through the cell membrane and into the cell. The target, forexample, can be a second intercellular protein that has catalyticactivity or a protein that facilitates the association of downstreamsignaling molecules with BFLP0169.

Determining the ability of the BFLP0169 protein to bind to or interactwith a BFLP0169 target molecule can be accomplished by one of themethods described above for determining direct binding. In oneembodiment, determining the ability of the BFLP0169 protein to bind toor interact with a BFLP0169 target molecule can be accomplished bydetermining the activity of the target molecule. For example, theactivity of the target molecule can be determined by detecting inductionof a cellular second messenger of the target (i.e. intracellular Ca²⁺,diacylglycerol, IP₃, etc.), detecting catalytic/enzymatic activity ofthe target an appropriate substrate, detecting the induction of areporter gene (comprising a BFLP0169-responsive regulatory elementoperatively linked to a nucleic acid encoding a detectable marker, e.g.,luciferase), or detecting a cellular response, for example, cellsurvival, cellular differentiation, or cell proliferation.

In yet another embodiment, an assay of the invention is a cell-freeassay comprising contacting a BFLP0169 protein or biologically-activeportion thereof with a test compound and determining the ability of thetest compound to bind to the BFLP0169 protein or biologically-activeportion thereof. Binding of the test compound to the BFLP0169 proteincan be determined either directly or indirectly as described above. Inone such embodiment, the assay comprises contacting the BFLP0169 proteinor biologically-active portion thereof with a known compound which bindsBFLP0169 to form an assay mixture, contacting the assay mixture with atest compound, and determining the ability of the test compound tointeract with a BFLP0169 protein, wherein determining the ability of thetest compound to interact with a BFLP0169 protein comprises determiningthe ability of the test compound to preferentially bind to BFLP0169 or abiologically-active portion thereof as compared to the known compound.

In still another embodiment, an assay is a cell-free assay comprisingcontacting BFLP0169 protein or a biologically-active portion thereofwith a test compound and determining the ability of the test compound tomodulate (e.g. stimulate or inhibit) the activity of the BFLP0169protein or a biologically-active portion thereof. Determining theability of the test compound to modulate the activity of BFLP0169 can beaccomplished, for example, by determining the ability of the BFLP0169protein to bind to a BFLP0169 target molecule by one of the methodsdescribed above for determining direct binding. In an alternativeembodiment, determining the ability of the test compound to modulate theactivity of BFLP0169 protein can be accomplished by determining theability of the BFLP0169 protein further modulate a BFLP0169 targetmolecule. For example, the catalytic/enzymatic activity of the targetmolecule on an appropriate substrate can be determined as describedabove.

In yet another embodiment, the cell-free assay comprises contacting theBFLP0169 protein or a biologically-active portion thereof with a knowncompound which binds BFLP0169 protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a BFLP0169 protein,wherein determining the ability of the test compound to interact with aBFLP0169 protein comprises determining the ability of the BFLP0169protein to preferentially bind to or modulate the activity of a BFLP0169target molecule.

The cell-free assays of the invention are amenable for use with both thesoluble form or the membrane-bound form of BFLP0169 protein. In the caseof cell-free assays comprising the membrane-bound form of BFLP0169protein, it may be desirable to utilize a solubilizing agent such thatthe membrane-bound form of BFLP0169 protein is maintained in solution.Examples of such solubilizing agents include non-ionic detergents suchas n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),N-dodecyl--N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO).

In more than one embodiment of the above assay methods of the invention,it may be desirable to immobilize either BFLP0169 protein or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to BFLP0169 protein, or interaction ofBFLP0169 protein with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided that adds a domain that allows one orboth of the proteins to be bound to a matrix. For example, GST-BFLP0169fusion proteins or GST-target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, that are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or BFLP0169 protein, and the mixture is incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described, supra. Alternatively,the complexes can be dissociated from the matrix, and the level ofBFLP0169 protein binding or activity determined using standardtechniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either theBFLP0169 protein or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated BFLP0169 protein ortarget molecules can be prepared from biotin-NHS (N-hydroxy-succinimide)using techniques well-known within the art (e.g., biotinylation kit,Pierce Chemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with BFLP0169 protein or target molecules, but whichdo not interfere with binding of the BFLP0169 protein to its targetmolecule, can be derivatized to the wells of the plate, and unboundtarget or BFLP0169 protein trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the BFLP0169 protein or targetmolecule, as well as enzyme-linked assays that rely on detecting anenzymatic activity associated with the BFLP0169 protein or targetmolecule.

In another embodiment, modulators of BFLP0169 protein expression areidentified in a method wherein a cell is contacted with a candidatecompound and the expression of BFLP0169 mRNA or protein in the cell isdetermined. The level of expression of BFLP0169 mRNA or protein in thepresence of the candidate compound is compared to the level ofexpression of BFLP0169 mRNA or protein in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof BFLP0169 mRNA or protein expression based upon this comparison. Forexample, when expression of BFLP0169 mRNA or protein is greater (i.e.,statistically significantly greater) in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of BFLP0169 mRNA or protein expression. Alternatively, whenexpression of BFLP0169 mRNA or protein is less (statisticallysignificantly less) in the presence of the candidate compound than inits absence, the candidate compound is identified as an inhibitor ofBFLP0169 mRNA or protein expression. The level of BFLP0169 mRNA orprotein expression in the cells can be determined by methods describedherein for detecting BFLP0169 mRNA or protein.

In yet another aspect of the invention, the BFLP0169 proteins can beused as “bait proteins” in a two-hybrid assay or three hybrid assay, toidentify other proteins that bind to or interact with BFLP0169(“BFLP0169-binding proteins” or “BFLP0169-bp”) and modulate BFLP0169activity. Such BFLP0169-binding proteins are also likely to be involvedin the propagation of signals by the BFLP0169 proteins as, for example,upstream or downstream elements of the BFLP0169 pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for BFLP0169 is fusedto a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a BFLP0169-dependenht complex,the DNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) that is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene that encodes the protein which interacts with BFLP0169.

The invention further pertains to novel agents identified by theaforementioned screening assays and uses thereof for treatments asdescribed herein.

The invention will be illustrated in the following non-limitingexamples.

EXAMPLE 1 Expression Patterns of Murine BFLP0169 Sequence inDisease-free, Lupus Nephritis Simulated Disease, and Rapamycin-treatedDiseased Mice

The expression of murine BFLP0169 sequences were examined in mice thatdeveloped lupus nephritis-like symptoms in the art-recognized NZB X NZWmurine model (see, e.g., Drake et al., Genetic analysis of the NZBcontribution to lupus-like autoimmune disease in (NZB×NZW)F1 mice, ProcNatl Acad Sci USA 91:4062-66, 1994; Finck et al., Interleukin 6 promotesmurine lupus in NZB/NZWF1 mice, J. Clin. Invest 94:585-91, 1994;Guglielmotti et al., Bindarit prolongs survival and reduces renal damageof NSB/W lupus mice. Clin. Exp. Rheumatol. 16:149, 1998; Yang et al.,Dietary conjugated linoleic acid protects against end stage disease ofsystemic lupus erythematosus in the NZB/W F1 mouse, Immunopharmacol.Immunotoxicol. 22:433-49, 2000. Expression in diseased mice was comparedto expression of the sequences in non-diseased mice of varying ages, andin mice whose lupus nephritis-like symptoms diminished followingtreatment with rapamycin or anti-B7 antibodies.

Mice were obtained from Jackson Laboratories at 6 to 8 weeks of age andaged on site. Data were obtained from kidneys of mice and harvested atthe indicated time point: C57BL/6 female mice at 8, and 32 weeks,FI(NZB×NZW) female mice 12, 25, and 42 weeks, mice treated withrapamycin at 42 and 55 weeks, mice treated with antibodies to B7.1 andB7.2 at 52 weeks. Each group contained three mice.

Rapamycin treated mice received 5 mg/kg rapamycin subcutaneous injection3 times per week for 8 weeks staring at 29 weeks of age. Control micereceived injections of vehicle (methyl cellulose) on the same schedule.Effectiveness of therapy was determined by normalization of proteinuriaand kidney histology (data not shown). Gene expression analysis waspreformed on mice sacrificed at the end of the treatment course (36weeks of age, data not shown), and at 42 weeks (6 weeks after treatment)and 55 weeks (20 weeks after treatment).

Mice treated with anti-B7 received 200 μg of anti-B7.1 (1G10F9monoclonal) and 200 μg of anti-B7.2 (GL1 monoclonal) by intra-peritonealinjections 3 times per week for two weeks starting at 29 weeks of age.Gene expression analysis was performed 21 weeks after treatment.

RNA Isolation and Hybridization to Oligonucleotide Arrays

Kidneys from both male and female mice were collected and snap frozenfor RNA isolation. One half each kidney was used. A longitudinal sectionof the left kidney and a cross section of the right kidney was used infor each individual animal.

Snap frozen mouse kidney tissue was homogenized using homogenizersuspended in RLT buffer plus 2ME for 30 to 45 seconds. Total RNA wasprepared using the Qiagen Midi Kit following the manufacturer'sprotocol. RNA was suspended in DEPC treated H2O and quantified by OD280.

cDNA was synthesized from 5 ug of total RNA using the Superscript Kit(BRL). cDNA was purified using phenol:cloroform:isoamyl alcohol(25:24:1) with a Phage lock gel tube following the Phage lock protocol.Supernatant was collected and cleaned up using EtOH. Sample wasresuspended in DEPC treated H2O.

In vitro T7 polymerase driven transcription reactions for synthesis andbiotin labeling of antisense cRNA. Qiagen RNeasy spin columnpurification used used to purify the cRNA. GeneChip hybridizationmixtures contained 15 ug fragmented cRNA, 0.5 mg/ml acetylated BSA, 0.1mg/ml herring sperm DNA, in 1×MES buffer in a total volume of 200 ul asper manufactures instructions. Reaction mixtures were hybridized for 16hr at 45° C. to Affymetrix Mu11KsubA and Mu11KsubB oligonucleotidearrays. The hybridization mixtures were removed and the arrays werewashed and stained with Streptavidin R-phycoerthrin (Molecular Probes)using GeneChip Fluidics Station 400 and scanned with a Hewlett PackardGeneArray Scanner following manufactures instructions. Fluorescent datawas collected and converted to gene specific difference average usingMicroArray Suite software.

Analysis of Oligonucleotide Array Data

An eleven member standard curve, comprised of gene fragments derivedfrom cloned bacterial and bacteriophage sequences were spiked into eachhybridization mixture at concentrations ranging from 0.5 pM to 150 pMrepresenting RNA frequencies of approximately 3.3 to 1000 parts permillion (ppm). The biotinylated standard curve fragments weresynthesized by T7-polymerase driven IVT reactions from plasmid-basedtemplates. The spiked biotinylated RNA fragments serve both as aninternal standard to assess chip sensitivity and as standard curve toconvert measured fluorescent difference averages from individual genesinto RNA frequencies in ppm as described by Hill et al.

Gene expression frequencies from each individual mouse kidney weremeasured and the expression data subjected to statistical analysis.Frequency values determined from individual measurements for a givengroup of mice were averaged. Genes whose frequencies differedsignificantly between C57B16 kidneys at 12 and 32 weeks of age wereclassified as changing as a result of the normal aging process, and notdue to a disease process.

Expression frequencies in young (disease-free), old (diseased), andeffectively treated old (disease-free) F1(NZB×NZW) mice and C57BL6control mice of oligonucleotide sequence identified on the AffymetrixMurine 11K chip by the qualifier aa002653_s_at are shown. This sequencerepresents an unknown mouse gene.

The results are shown in FIG. 1. Shown is a histogram showing geneexpression levels in kidneys from the indicated mice. Expression levelsof BFLP0169 do not vary significantly between C57BL/6 kidneys at 12weeks of age and kidney at 32 weeks of age, indicating that expressionlevels do not increase with age in kidneys of non-diseased mice. In(NXB×NZW)F1 kidneys, the gene is expressed at normal levels prior todisease onset (12 weeks of age). As the mice age and disease progresses,increasing expression levels are observed at 25 weeks, 36 weeks (datanot shown for 36 weeks), and 42 weeks. By 55 weeks of age, the mice havedied due to kidney failure. Mice treated with rapamycin for 8 weeks withtreatment starting at 29 weeks of age, remain healthy past 55 weeks ofage. Kidneys of mice that have received effective therapy (eitherrapamycin therapy or anti-B7 therapy) express normal levels of BFLP0169,and these normal levels persist in asymptomatic kidney 20 weeks aftercessation of rapamycin therapy and 15 weeks after cessation of anti-B7therapy. The observation that expression levels return to normal whenkidney function is normal indicates that elevated levels are related to,and diagnostic of, disease progression. Blocking the function of thesegenes may inhibit or retard disease progression. Expression levels mayalso to used to assess and compare effectiveness of various therapeuticinterventions.

EXAMPLE 2 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the L at position 78 of the BFLP0169 sequence shown in Table 2has been replaced by a V, which is shown in bold font.

(SEQ ID NO:3) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKEVGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 3 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the L at position 198 of the BFLP0169 sequence shown in Table 2has been replaced by an 1, which is shown in bold font.

(SEQ ID NO:4) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNIMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 4 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the R at position 267 of the BFLP0169 sequence shown in Table 2has been replaced by a K, which is shown in bold font.

(SEQ ID NO:5) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDKIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 5 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the Q at position 355 of the BFLP0169 sequence shown in Table 2has been replaced by an N, which is shown in bold font.

(SEQ ID NO:6) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAINLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 6 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the H at position 446 of the BFLP0169 sequence shown in Table 2has been replaced by an R, which is shown in bold font.

(SEQ ID NO:7) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPRLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 7 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the L at position 553 of the BFLP0169 sequence shown in Table 2has been replaced by an I, which is shown in bold font.

(SEQ ID NO:8) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMILHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 8 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the W at position 634 of the BFLP0169 sequence shown in Table 2has been replaced by a F, which is shown in bold font.

(SEQ ID NO:9) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVFTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 9 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the N at position 749 of the BFLP0169 sequence shown in Table 2has been replaced by a D, which is shown in bold font.

(SEQ ID NO:10) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILDLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 10 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the V at position 838 of the BFLP0169 sequence shown in Table 2has been replaced by a M, which is shown in bold font.

(SEQ ID NO:11) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVMSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 11 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the G at position 958 of the BFLP0169 sequence shown in Table 2has been replaced by a T, which is shown in bold font.

(SEQ ID NO:12) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLTMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 12 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the K at position 1084 of the BFLP0169 sequence shown in Table 2has been replaced by a R, which is shown in bold font.

(SEQ ID NO:13) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVRSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 13 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the A at position 1152 of the BFLP0169 sequence shown in Table 2has been replaced by a S, which is shown in bold font.

(SEQ ID NO:14) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPSSVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 14 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the I at position 1247 of the BFLP0169 sequence shown in Table 2has been replaced by a V, which is shown in bold font.

(SEQ ID NO:15) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVVALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 15 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the K at position 1331 of the BFLP0169 sequence shown in Table 2has been replaced by a R, which is shown in bold font.

(SEQ ID NO:16) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMRQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 16 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the C at position 1449 of the BFLP0169 sequence shown in Table 2has been replaced by a Y, which is shown in bold font.

(SEQ ID NO:17) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLAYLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 17 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the D at position 1542 of the BFLP0169 sequence shown in Table 2has been replaced by a Q, which is shown in bold font.

(SEQ ID NO:18) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPSSVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAAQTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYFRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

EXAMPLE 18 A Variant of the Human BFLP0169 Polypeptide Sequence Shown inTable 2

A polypeptide sequence varying by one amino acid from the BFLP0169 aminoacid sequence presented in Table 2 is shown below. For the sequenceshown, the F at position 1706 of the BFLP0169 sequence shown in Table 2has been replaced by a H, which is shown in bold font.

(SEQ ID NO:18) MIRKSKITSVLSFCRSSRELWTILLGRSALRELSQIEAELNKHWRRLLEGLSYYKPPSPSSAEKVKANKDVASPLKELGLRISKFLGLDEEQSVQLLQCYLQEDYRGTRDSVKTVLQDERQSQALILKIADYYYEERTCILRCVLHLLTYFQDERHPYRVEYADCVDKLEKELVSKYRQQFEELYKTEAPTWETHGNLMTERQVSRWFVQCLREQSMLLEIIFLYYAYFEMAPSDLLVLTKMFKEQGFGSRQTNRHLVDETMDPFVDRIGYFSALILVEGMDIESLHKCALDDRRELHQFAQDGLICQDMDCLMLTFGDIPHHAPVLLAWALLRHTLNPEETSSVVRKIGGTAIQLNVFQYLTRLLQSLASGGNDCTTSTACMCVYGLLSFVLTSLELHTLGNQQDIIDTACEVLADPSLPELFWGTEPTSGLGIILDSVCGMFPHLLSPLLQLLRALVSGKSTAKKVYSFLDKMSFYNELYKHKPHDVISHEDGTLWRRQTPKLLYPLGGQTNLRIPQGTVGQVMLDDRAYLVRWEYSYSSWTLFTCEIEMLLHVVSTADVIQHCQRVKPIIDLVHKVISTDLSIADCLLPITSRIYMLLQRLTTVISPPVDVIASCVNCLTVLAARNPAKVWTDLRHTGFLPFVAHPVSSLSQMISAEGMNAGGYGNLLMNSEQPQGEYGVTIAFLRLITTLVKGQLGSTQSQGLVPCVMFVLKEMLPSYHKWRYNSHGVREQIGCLILELIHAILNLCHETDLHSSHTPSLQFLCICSLAYTEAGQTVINIMGIGVDTIDMVMAAQPRSDGAEGQGQGQLLIKTVKLAFSVTNNVIRLKPPSNVVSPLEQALSQHGAHGNNLIAVLAKYIYHKHDPALPRLAIQLLKRLATVAPMSVYACLGNDAAAIRDAFLTRLQSKIEDMRIKVMILEFLTVAVETQPGLIELFLNLEVKDGSDGSKEFSLGMWSCLHAVLELIDSQQQDRYWCPPLLHRAAIAFLHALWQDRRDSAMLVLRTKPKFWENLTSPLFGTLSPPSETSEPSILETCALIMKIICLEIYYVVKGSLDQSLKDTLKKFSIEKRFAYWSGYVKSLAVHVAETEGSSCTSLLEYQMLVSAWRMLLIIATTHADIMHLTDSVVRRQLFLDVLDGTKALLLVPASVNCLRLGSMKCTLLLILLRQWKRELGSVDEILGPLTEILEGVLQADQQLMEKTKAKVFSAFITVLQMKEMKVSDIPQYSQLVLNVCETLQEEVIALFDQTRHSLALGSATEDKDSMETDDCSRSRHRDQRDGVCVLGLHLAKELCEVDEDGDSWLQVTRRLPILPTLLTTLEVSLRMKQNLHFTEATLHLLLTLARTQQGATAVAGAGITQSICLPLLSVYQLSTNGTAQTPSASRKSLDAPSWPGVYRLSMSLMEQLLKTLRYNFLPEALDFVGVHQERTLQCLNAVRTVQSLACLEEADHTVGFILQLSNFMKEWHFHLPQLMRDIQVNLGYLCQACTSLLHSRKMLQHYLQNKNGDGLPSAVAQRVQRPPSAASAAPSSSKQPAADTEASEQQALHTVQYGLLKILSKTLAALRHFTPDVCQILLDQSLDLAEYNFLFALSFTTPTFDSEVAPSFGTLLATVNVALNMLGELDKKKEPLTQAVGLSTQAEGTRTLKSLLMFTMENCFYLLISQAMRYLRDPAVHPRDKQRMKQELSSELSTLLSSLSRYHRRGAPSSPATGVLPSPQGKSTSLSKASPESQEPLIQLVQAFVRH MQR

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1. An isolated nucleic acid molecule encoding a polypeptide comprisingthe amino acid sequence selected from the group, consisting of SEQ IDNOs:2 through
 19. 2. A vector comprising the nucleic acid molecule ofclaim
 1. 3. An isolated cell comprising the vector of claim
 2. 4. Amethod of producing a polypeptide, said method comprising culturing acell comprising the nucleic acid molecule of claim 1 under conditionsallowing for expression of the polypeptide encoded by said nucleic acidmolecule.
 5. The isolated nucleic acid molecule of claim 1 wherein thenucleic acid molecule encodes the amino acid sequence set forth in SEQID NO:2.
 6. The isolated nucleic acid molecule of claim 1 wherein thenucleic acid molecule encodes the amino acid sequence set forth in SEQID NO:3.
 7. The isolated nucleic acid molecule of claim 1 wherein thenucleic acid molecule encodes the amino acid sequence set forth in SEQID NO:4.
 8. The isolated nucleic acid molecule of claim 1 wherein thenucleic acid molecule encodes the amino acid sequence set forth in SEQID NO:5.
 9. The isolated nucleic acid molecule of claim 1 wherein thenucleic acid molecule encodes the amino acid sequence set forth in SEQID NO:6.
 10. The isolated nucleic acid molecule of claim 1 wherein thenucleic acid molecule encodes the amino acid sequence set forth in SEQID NO:7.
 11. The isolated nucleic acid molecule of claim 1 wherein thenucleic acid molecule encodes the amino acid sequence set forth in SEQID NO:8.
 12. The isolated nucleic acid molecule of claim 1 wherein thenucleic acid molecule encodes the amino acid sequence set forth in SEQID NO:9.
 13. The isolated nucleic acid molecule of claim 1 wherein thenucleic acid molecule encodes the amino acid sequence set forth in SEQID NO:10.
 14. The isolated nucleic acid molecule of claim 1 wherein thenucleic acid molecule encodes the amino acid sequence of SEQ ID NO:11.15. The isolated nucleic acid molecule of claim 1 wherein the nucleicacid molecule encodes the amino acid sequence set forth in SEQ ID NO:12.16. The isolated nucleic acid molecule of claim 1 wherein the nucleicacid molecule encodes the amino acid sequence set forth in SEQ ID NO:13.17. The isolated nucleic acid molecule of claim 1 wherein the nucleicacid molecule encodes the amino acid sequence set forth in SEQ ID NO:14.18. The isolated nucleic acid molecule of claim 1 wherein the nucleicacid molecule encodes the amino acid sequence set forth in SEQ ID NO:15.19. The isolated nucleic acid molecule of claim 1 wherein the nucleicacid molecule encodes the amino acid sequence set forth in SEQ ID NO:16.20. The isolated nucleic acid molecule of claim 1 wherein the nucleicacid molecule encodes the amino acid sequence set forth in SEQ ID NO:17.21. The isolated nucleic acid molecule of claim 1 wherein the nucleicacid molecule encodes the amino acid sequence set forth in SEQ ID NO:18.22. The isolated nucleic acid molecule of claim 1 wherein the nucleicacid molecule encodes the amino acid sequence set forth in SEQ ID NO:19.